KR20200131279A - IL-13 receptor alpha 2 (IL13RA2) chimeric antigen receptor for tumor specific T cell immunotherapy - Google Patents

Abstract

Some embodiments of the methods and compositions provided herein include chimeric antigen receptors (CARs) that specifically bind to the human extracellular domain of the IL-13 alpha 2 (IL13Ra2) receptor, cells containing such CARs, and cancer cells, such as It relates to a method of cell-based immunotherapy that targets cells of solid tumors.

Description

IL-13 receptor alpha 2 (IL13RA2) chimeric antigen receptor for tumor specific T cell immunotherapy

<Cross reference to related applications>

This application claims priority to U.S. Provisional Application No. 62/643055, filed March 14, 2018, entitled "IL-13 Receptor Alpha 2 (IL13RA2) Chimeric Antigen Receptor for Tumor Specific T Cell Immunotherapy" , Which is expressly incorporated herein by reference in its entirety.

<Reference to the sequence list>

This application is filed with a sequence listing in electronic format. The sequence listing is provided as a file with the file name SCRI169WOSEQLIST generated on March 12, 2019, which is approximately 62 Kb in size. Information in electronic form of the Sequence Listing is expressly incorporated herein by reference in its entirety.

<Technical field of the invention>

Some embodiments of the methods and compositions provided herein include chimeric antigen receptors (CARs) that specifically bind to the human extracellular domain of the IL-13 alpha 2 (IL13Ra2) receptor, cells containing such CARs, and cancer cells, such as It relates to a method of cell-based immunotherapy that targets cells of solid tumors.

<Background of the invention>

Despite significant advances in understanding of brain cancer, mortality rates have remained consistent over the past decade and new innovative therapies are urgently needed. To date, T cell immunotherapy has emerged as a promising cancer therapy supported by prominent clinical data reporting complete remission in patients with B cell malignancies following administration of T cell CARs targeting CD19. However, there remains a need for additional and improved T cell immunotherapy.

<Summary of the invention>

Some embodiments of the methods and compositions provided herein comprise a nucleic acid encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises a ligand binding domain that binds and/or interacts with the IL-13 alpha 2 (IL13Rα2) receptor; A polypeptide spacer between the ligand binding domain and the transmembrane domain; Transmembrane domain; And an intracellular signaling region.

In some embodiments, the ligand binding domain comprises: a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 20, or a conservative variant thereof; Heavy chain complementarity determining region 2 (CDR2) having the amino acid sequence of SEQ ID NO: 21, or a conservative variant thereof; And/or heavy chain complementarity determining region 3 (CDR3) having the amino acid sequence of SEQ ID NO: 22, or a conservative variant thereof.

In some embodiments, the ligand binding domain comprises: a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:23, or a conservative variant thereof; Light chain complementarity determining region 2 (CDR2) having the amino acid sequence of SEQ ID NO: 24, or a conservative variant thereof; And/or the amino acid sequence of SEQ ID NO: 25, or a light chain complementarity determining region 3 (CDR3) having a conservative variant thereof.

In some embodiments, the ligand binding domain comprises a heavy chain variable (VH) domain comprising a polypeptide having at least 90% identity to the amino acid sequence of SEQ ID NO:18.

In some embodiments, the ligand binding domain comprises a light chain variable (VL) domain comprising a polypeptide having at least 90% identity to the amino acid sequence of SEQ ID NO:19.

In some embodiments, the ligand binding domain comprises: a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:20; Heavy chain CDR2 having the amino acid sequence of SEQ ID NO:21; And/or a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:22.

In some embodiments, the ligand binding domain comprises: a light chain CDR1 having the amino acid sequence of SEQ ID NO:23; Light chain CDR2 having the amino acid sequence of SEQ ID NO:24; And/or a light chain CDR3 having the amino acid sequence of SEQ ID NO:25.

In some embodiments, the ligand binding domain comprises a VH domain comprising a polypeptide having the amino acid sequence of SEQ ID NO:18.

In some embodiments, the ligand binding domain comprises a VL domain having the amino acid sequence of SEQ ID NO:19.

In some embodiments, the ligand binding domain is an antibody fragment, such as an antigen-binding fragment.

In some embodiments, the ligand binding domain is a single chain variable fragment (scFv). In some embodiments, the scFv comprises a VL-VH orientation. In some embodiments, the single chain variable fragment (scFv) comprises a sequence having at least 95% identity to the nucleotide sequence of SEQ ID NO:61. In some embodiments, the single chain variable fragment (scFv) comprises the nucleotide sequence of SEQ ID NO:61.

In some embodiments, the spacer comprises the amino acid sequence of X ₁ PPX ₂ P.

In some embodiments, the spacer region comprises a portion of the hinge region of a human antibody or a modified variant thereof.

In some embodiments, the spacer is 15 or fewer amino acids, but 1 or 2 or more amino acids.

In some embodiments, the spacer comprises, consists of, or consists essentially of a sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:09. In some embodiments, the spacer comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:09 or a conservative substitution thereof.

In some embodiments, the spacer comprises, consists of, or consists essentially of an IgG4 hinge spacer (S).

In some embodiments, the spacer comprises, consists of, or consists essentially of an IgG4 hinge-CH3 spacer (M).

In some embodiments, the spacer comprises, consists of, or consists essentially of an IgG4 hinge-CH2 (L234D, N297A)-CHE spacer (L).

In some embodiments, the intracellular signaling region is the signaling domain of CD27, CD28, 4-1BB, OX-40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, NKG2C and B7-H3 And a primary and co-stimulatory signaling domain optionally containing all or part of CD3 zeta in combination with a co-stimulatory domain selected from the group consisting of combinations thereof.

In some embodiments, the intracellular signaling region comprises a signaling portion of CD3 zeta and a signaling portion of 4-1BB.

In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain (CD28tm).

Some embodiments also include nucleic acids encoding marker sequences. In some embodiments, the marker sequence is a truncated receptor, optionally EGFRt, HER2t or CD19t.

Some embodiments also include a dihydrofolate reductase transgene configured for methotrexate selection. In some embodiments, the dihydrofolate reductase transgene is a dihydrofolate reductase double mutant (DHFRdm). In some embodiments, the dihydrofolate reductase double mutant comprises amino acid mutations of L22F and F31S.

Some embodiments of the methods and compositions provided herein include an expression vector comprising the nucleic acid of any of the embodiments provided herein. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a lentiviral or adenovirus vector.

Some embodiments of the methods and compositions provided herein include chimeric antigen receptor (CAR) polypeptides encoded by the nucleic acids of any one of the embodiments provided herein.

Some embodiments of the methods and compositions provided herein include host cells comprising the nucleic acid of any of the embodiments provided herein.

In some embodiments, the host cell is a CD8+ T cytotoxic lymphocyte cell selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, and bulk CD8+ T cells. In some embodiments, the CD8+ cytotoxic T lymphocyte cells are central memory T cells, wherein the central memory T cells are positive for CD45RO+, CD62L+, and CD8+.

In some embodiments, the host cell is a CD4+ T helper lymphocyte cell selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells. In some embodiments, the CD4+ helper lymphocyte cells are naive CD4+ T cells, wherein the naive CD4+ T cells are positive for CD45RA+, CD62L+, and CD4+ and negative for CD45RO.

In some embodiments, the host cell is a precursor T cell.

In some embodiments, the host cell is a hematopoietic stem cell.

Some embodiments of the methods and compositions provided herein include pharmaceutical compositions comprising a host cell of any one of the embodiments provided herein and a pharmaceutically acceptable excipient.

Some embodiments of the methods and compositions provided herein include a method of making a host cell of any one of the embodiments provided herein comprising: introducing into the cell the nucleic acid of any of the embodiments provided herein; Culturing the cells in conditions sufficient for the cells to expand in the presence of an anti-CD3 antibody and/or anti CD28 antibody and at least one homeostatic cytokine; And selecting cells as a selection reagent; Wherein the selection reagent is configured to selectively enrich cells transduced with a nucleic acid or vector.

In some embodiments, the cell is a lymphocyte. In some embodiments, the lymphocyte has a CD45RA-, CD45RO+, and CD62L+ phenotype. In some embodiments, the lymphocyte is CD8+ or CD4+.

In some embodiments, the reagent of choice is methotrexate.

In some embodiments, the cytokine is IL-15, Il-7 and/or Il-21.

Some embodiments also include introducing a second nucleic acid into the host cell, wherein the second nucleic acid encodes a marker protein. In some embodiments, the marker protein is EGFRt.

Some embodiments of the methods and compositions provided herein include a host cell of any of the embodiments provided herein for use in a medicament or for use in the treatment or inhibition of a cancer or solid tumor expressing the IL-13α2 receptor. . In some embodiments, the cancer comprises brain cancer. In some embodiments, the cancer is a glioma or glioblastoma tumor. In some embodiments, the cancer is glioblastoma polymorphic (GBM). In some embodiments, the cancer is a glioma.

Some embodiments of the methods and compositions provided herein include methods of treating, inhibiting or alleviating cancer in a subject comprising administering to a subject in need thereof the host cell of any one of the embodiments provided herein. . In some embodiments, the cancer is an IL13Rα-positive malignant tumor. In some embodiments, the cancer is brain cancer. In some embodiments, the cancer is a glioma or glioblastoma tumor. In some embodiments, the cancer is a glioma. In some embodiments, the cancer is glioblastoma polymorphic (GBM). In some embodiments, the subject is a mammal. In some embodiments, the subject is human.

Some embodiments also include administering an additional therapy selected from chemotherapy and radiation therapy. In some embodiments, the chemotherapeutic drug is electrochemotherapy, alkylating agents, antimetabolites (e.g., 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine (gel Roda®), cladribine, cloparabine, cytarabine (Ara-C®), phloxuridine, fludarabine, gemcitabine (Gemza®), hydroxyurea, methotrexate, pemetrexide (Alymta®), Pentostatin, and thioguanine), anti-tumor antibiotics, topoisomerase inhibitors, mitosis inhibitors, corticosteroids, DNA intercalating agents, or checkpoint inhibitors (checkpoint kinases CHK1, CHK2).

1 depicts a schematic diagram of the components used for construction of certain embodiments of IL-13Rα2-targeting CARs, and embodiments of IL-13Rα2-targeting CARs. The IL-13Rα2 specific CAR included: specific for the extracellular epitope of the IL-13 alpha 2 receptor (IL-13Rα2) derived from either antibody'hu08' (Ab01) or'hu07' (Ab02). One of numerous single-chain variable fragments (scFv) that are recognized as and contain the VH and VL domains with a linker between them; One of the various spacer domains (denoted as “S”, “M” and “L”); A transmembrane domain derived from human CD28 (CD28tm); A costimulatory domain derived from human 4-1BB; CD3ζ-derived signaling domain; T2A ribosome skip sequence; And a truncated EGFR (EGFRt) transduction marker; And a dihydrofolate reductase double mutant (DHFRdm) transgene, optionally configured for additional T2A ribosomal skip sequence and methotrexate selection.
FIG. 2A depicts flow cytometric analysis of EGFRt expression in CD8+ T cells (upper panel) or CD4+ T cells (lower panel) transduced with the various CARs shown in FIG. 1. From top row to bottom row of each panel: T cell mock; T cells with IL-13Ra2 Ab01 VL-VH spacer S CAR; T cells with IL-13Ra2 Ab01 VH-VL spacer S CAR; T cells with IL-13Ra2 Ab02 VL-VH spacer S CAR; And T cells with IL-13Ra2 Ab02 VH-VL spacer S CAR.
2B shows a graph of cytokine production (IL-2, IFN-γ, TNF-α) from CD8+ T cells transduced with various CARs and incubated with the indicated target cells. The key is T cell mock; T cells with IL-13Ra2 Ab01 VL-VH spacer S CAR; T cells with IL-13Ra2 Ab01 VH-VL spacer S CAR; T cells with IL-13Ra2 Ab02 VL-VH spacer S CAR; And IL-13Ra2 Ab02 VH-VL spacer S CAR.
FIG. 2C depicts a graph of cytokine production (IL-2, IFN-γ, TNF-α) from CD4+ T cells transduced with various CARs and incubated with the indicated target cells. The key is T cell mock; T cells with IL-13Ra2 Ab01 VL-VH spacer S CAR; T cells with IL-13Ra2 Ab01 VH-VL spacer S CAR; T cells with IL-13Ra2 Ab02 VL-VH spacer S CAR; And IL-13Ra2 Ab02 VH-VL spacer S CAR.
3A is a multi-parameter flow cytometry analysis of T cells derived from healthy donors and transduced with IL-13Rα2 hu08 CAR, with histogram quadrants plotted based on control staining (top and middle panels); And flow cytometric analysis of EGFRt expression in CD8+ T cells transduced with CAR (lower panel). CARs indicated included: mock; IL-13Ra2 Ab01 VL-VH spacer-S CAR; IL-13Ra2 Ab01 VL-VH spacer-M CAR; IL-13Ra2 Ab01 VL-VH spacer-L CAR; IL-13Ra2 Ab01 VH-VL spacer-S CAR; IL-13Ra2 Ab01 VH-VL spacer-M CAR; And IL-13Ra2 Ab01 VH-VL spacer-L CAR.
3B depicts the assay for cytokine production and cytolytic activity of CD8+ T cells containing CAR after co-culture with certain indicated target cells. CARs indicated included: mock; IL-13Ra2 Ab01 VL-VH spacer-S CAR; IL-13Ra2 Ab01 VL-VH spacer-M CAR; IL-13Ra2 Ab01 VL-VH spacer-L CAR; IL-13Ra2 Ab01 VH-VL spacer-S CAR; IL-13Ra2 Ab01 VH-VL spacer-M CAR; And IL-13Ra2 Ab01 VH-VL spacer-L CAR.
Figure 4A shows a graph of total flux (photons/second) as a measure of tumor burden (y-axis) over time (days after tumor inoculation, x-axis) for mice treated with: vehicle, mock Cells, 2 x 10 ⁶ T cells containing anti-IL-13Rα2 CAR; 1 x 10 ⁶ T cells containing anti-IL-13Rα2 CAR; Or 0.5 x 10 ⁶ T cells containing anti-IL-13Rα2 CAR. The anti-IL-13Rα2 CAR was an IL-13Ra2 Ab01 VL-VH spacer-S CAR.
Figure 4B depicts Kaplan-Meier survival curves for mice treated with increasing doses of T cells expressing anti-IL-13Rα2 CAR (IL-13Ra2 Ab01 VL-VH spacer-S CAR). .
5A shows a graph of total flux (photons/sec) as a measure of tumor burden (y-axis) over time (days after tumor inoculation, x-axis) for mice treated with: mock cells; 2 x 10 ⁶ T cells containing anti-IL-13Rα2 CAR containing short spacers; 2 x 10 ⁶ T cells containing anti-IL-13Rα2 CAR containing an intermediate spacer; 2 x 10 ⁶ T cells containing anti-IL-13Rα2 CAR containing long spacers.
5B depicts Kaplan-Meier survival curves for mice treated with T cells expressing anti-IL-13Rα2 CAR containing long spacers, intermediate spacers or short spacers.
5C depicts the area under the curve (AUC) analysis of bioluminescence data for mice treated with T cells expressing anti-IL-13Rα2 CAR containing long spacers, medium spacers or short spacers. The mean AUC between IL13Rα2 Ab01 spatial variant CAR T-cells or mock T-cells was compared. *, P<0.05. **, P<0.01. ****, P<0.0001.

<Detailed explanation>

Some embodiments of the methods and compositions provided herein include IL13Ra2-targeting chimeric antigen receptor (CAR), such as second generation IL-13Rα2-specific CARs, cells containing such CARs, nucleic acids encoding such CARs, and related therapeutic methods. And uses thereof. Among the provided CARs are those that have certain combinations of components or domains, such as those resulting from optimization of CAR function. In some aspects, IL13Ra2 CAR-targeted T cell therapeutics can also serve as an alternative or supplement to current IL-13Rα2-specific and nonspecific cancer treatments, such as combination therapy.

IL-13Rα2 has previously been shown to be abundant in metastatic or late BLBC (Papageorgis et al. Breast Cancer Research, 2015; 17 (1); the entirety of which is expressly incorporated herein by reference). Based on publicly available data, a correlation was made between progression-free survival-based likelihood and high levels of IL-13Rα2. A subtype of BLBC, which tends to spread rapidly to the lungs, was observed to have high IL-13Rα2 levels. IL-13Rα2 has also been shown to stimulate human glioma cell growth and metastasis via the Src/PI3K/Akt/mTOR signaling pathway. (Tu et al. Tumor Biol. 2016 Nov; 37(11):14701-14709; the entirety of which is expressly incorporated herein by reference). IL13Ra2 targeted therapies such as chimeric receptor-based therapies have been described (eg, Brown et al. Clin Cancer Res 2015; Brown et al. N Engl J Med 2016); Brown et al. Mol Ther 2017]; see WO 2014072888-A1, describes anti-IL-13 receptor alpha 2 (IL-13-Ra2) antibodies and antibody-drug conjugates for cancer treatment, each of which is expressly incorporated herein by reference in its entirety Included). Among these therapies are those based on or comprising a mutant IL13 (eg, E13Y)-based binding or antigen recognition domain, such as zetacaine.

In some embodiments, provided CARs and associated methods and uses are in the context of glioblastoma or a model thereof, in combination with certain other constituents, such as certain spacer domain curatives, of a CAR containing a particular binding domain and/or in vivo It is based in part on the observations described herein showing anticancer effects. In some aspects, provided compositions, methods and uses are used for the treatment, inhibition or prevention of glioblastoma and/or other IL-13Rα2-positive malignancies. In some aspects, provided CARs described herein differ from other CARs in various aspects of the IL-13Rα2 epitope specifically recognized by their molecular structure, such as binding domains, and targeted by the CARs provided herein, such as one or more characteristics. Do.

Embodiments provided are the observations herein, such as, to a greater extent, anti-tumor in vitro activity of provided CAR-T cells against glioblastoma cell lines and tumor growth in xenograft models compared to different CARs with different molecular structures in some aspects. It is based in part on what has been demonstrated in the preclinical studies described herein, demonstrating the ability to reduce. In light of these findings, the commercial use of compositions comprising these CARs in immunotherapy against brain tumors and other IL-13Rα2-positive types of cancer is contemplated.

<Definition>

As used herein, “nucleic acid” or “nucleic acid molecule” has its ordinary and general meaning when read in light of the specification, for example polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), Oligonucleotides, fragments produced by polymerase chain reaction (PCR), and fragments produced by any of ligation, cleavage, endonuclease action, and exonuclease action, but are not limited thereto. Does not. Nucleic acid molecules can be composed of monomers that are naturally-occurring nucleotides (such as DNA and RNA), or analogs of naturally-occurring nucleotides (e.g., enantiomeric forms of naturally-occurring nucleotides), or a combination of both. Modified nucleotides may have alterations in sugar moieties and/or pyrimidine or purine base moieties. Sugar modifications include, for example, replacing one or more hydroxyl groups with halogen, alkyl groups, amine and azido groups, or sugars may be functionalized with ethers or esters. Moreover, the entire sugar moiety can be replaced by a sterically and electronically similar structure, such as aza-sugar and carbocyclic sugar analogs. Examples of modifications of the base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substituents. Nucleic acid monomers may be linked by phosphodiester linkages or analogs of such linkages. Phosphodiester linkage analogs include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilideate, phosphoramidate, etc. Include. The term “nucleic acid molecule” also includes so-called “peptide nucleic acids”, which include naturally-occurring or modified nucleic acid bases attached to the polyamide backbone. Nucleic acids can be single stranded or double stranded. In some alternatives, a nucleic acid sequence encoding a protein is provided. In some alternatives, the nucleic acid is RNA or DNA.

As used herein, “chimeric antigen receptor” has its ordinary and general meaning when read in light of the specification, for example, by binding to a molecule associated with a disease or disorder and via a spacer domain, a cell, such as a T cell, or other It may include, but is not limited to, a synthetically designed receptor comprising one or more intracellular signaling domains of the receptor, such as a ligand binding domain of an antibody or other protein sequence linked to a costimulatory domain. Chimeric receptors may also be referred to as artificial cell receptors or T cell receptors, chimeric cell receptors or T cell receptors, chimeric immunoreceptors, and/or chimeric antigen receptors (CARs). These receptors can be used to implant the specificity of a monoclonal antibody or binding fragment thereof into a cell, preferably a T-cell, with the transfer of its coding sequence facilitated by a viral vector, such as a retroviral vector or a lentiviral vector. have. In some cases, the CAR may be a genetically engineered T cell receptor designed to redirect T cells to target cells expressing specific cell-surface antigens. T cells can be removed from the subject and modified to express receptors that may be antigen-specific by a process called adoptive cell migration. T cells are reintroduced into the patient, where they can recognize and target the antigen. CAR is also an engineered receptor capable of implanting any specificity into immune receptor cells. The term chimeric antigen receptor or “CAR” is contemplated by some investigators to include an antibody or antibody fragment, preferably an antigen binding fragment of an antibody, a spacer, a signaling domain, and a transmembrane region. Due to the surprising effect of modifying the different components or domains of the CARs described herein, such as epitope binding regions (e.g., antibody fragments, scFvs, or portions thereof), spacers, transmembrane domains, and/or signaling domains). In other words, the components of the CAR are often distinguished throughout the present disclosure in terms of independent elements. For example, mutations in different elements of the CAR can lead to a stronger binding affinity for a specific epitope or antigen.

CARs are grafted into T cells with transfer of their coding sequences facilitated by the vector to the specificity of a monoclonal antibody or binding fragment thereof or scFv. In order to use CAR as a therapy for a subject in need thereof, a technique called adoptive cell migration is used to remove T cells from the subject and modify them to express an antigen-specific CAR. T cells capable of recognizing and targeting the antigen are then reintroduced into the patient.

In some embodiments, a transmembrane domain is a region of a transmembrane protein that is hydrophobic that may exist in a bilayer of cells to immobilize proteins embedded in a biological membrane. Without limitation, the topology of the transmembrane domain may be a transmembrane alpha helix. In some alternatives to the chimeric antigen receptor, the chimeric antigen receptor comprises a sequence encoding a transmembrane domain. In some alternatives, the transmembrane domain is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 amino acids in length or CD28 transmembrane sequences or fragments thereof that are of length within a range defined by any two of the aforementioned lengths. In some alternatives, the CD28 transmembrane sequence or fragment thereof comprises 28 amino acids in length.

In some embodiments, a signaling domain, such as a primary signaling domain or costimulatory domain, is an intracellular or cytoplasmic domain of a protein or receptor protein capable of interacting with and relaying a signal or participating in the relay of a signal. Includes. In some aspects, such interactions can occur through an effector molecule or an intracellular domain that communicates with an effector protein through a specific protein-protein or protein-ligand interaction, which in turn can send a signal along the signal chain to its destination. have. In some embodiments, the signaling domain comprises a costimulatory domain. In some aspects, the co-stimulatory domain is a response, such as a T-cell effector response, such as an immune response, activation, proliferation, differentiation, cytokine, in addition to the primary signal provided by, for example, the CD3 zeta chain of the TCR/CD3 complex. It includes a signaling moiety that provides a signal to T-cells that enhance secretion, cytolytic activity, perforin and/or granzyme activity, and the like. In some embodiments, the intracellular signaling domain and/or costimulatory domain is CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-l (LFA-l), CD2, CD7, All or part of LIGHT, NKG2C and/or B7-H3, and/or a ligand that specifically binds to CD83.

As used herein, “antibody” has its mediocre and general meaning when read in light of the specification, for example produced by plasma cells used by the immune system to identify and neutralize foreign substances such as bacteria and viruses. Large Y-shaped protein, but is not limited thereto. In some contexts, the term antibody refers to an antigen binding fragment of an antibody. The antibody protein comprises four polypeptide chains; It may contain two identical heavy chains and two identical light chains linked by disulfide bonds. Each chain consists of a structural domain called an immunoglobulin domain. These domains may contain 70-110 amino acids and are classified into different categories according to their size and function. The chimeric antigen receptor may comprise a ligand binding domain, which comprises an antibody fragment, preferably an antigen binding fragment. In some alternatives, a nucleic acid encoding a chimeric antigen receptor (CAR) is provided, wherein the nucleic acid is a) a first polynucleotide encoding a ligand binding domain, wherein the ligand binding domain binds to an IL-13 alpha 2 (IL13Rα2) receptor And/or interacts with, b) a second polynucleotide encoding a polypeptide spacer of sufficient length to allow the ligand binding domain to interact with the IL-13 alpha 2 (IL13Rα2) receptor, c) encoding a transmembrane domain A third polynucleotide, and d) a fourth polynucleotide encoding an intracellular signaling domain. In some alternatives, the ligand binding domain is an antibody fragment.

As used herein, “single chain variable fragment” or scFv has its ordinary and general meaning when read in light of the specification, for example heavy chain (VH) and light chain (VL) of immunoglobulins linked together by short linker peptides. ) May include a fusion protein including the variable region, but is not limited thereto. Without limitation, the linker may comprise glycine for flexibility and hydrophilic amino acids for solubility, such as serine or threonine. The linker may connect the N-terminus of VH to the C-terminus of VL, or may connect the C-terminus of VH to the N-terminus of VL. In some alternatives, the ligand binding domain present in the CAR is a single chain variable fragment (scFv). In some alternatives, the scFv domain present in the CAR is specific for the IL-13 alpha 2 (IL13Rα2) receptor present in tumor cells.

In some embodiments, the compositions, cells and vectors comprise a marker sequence or a nucleic acid encoding the same, which may, for example, comprise a protein that serves as a label for the cell. In some alternatives of the cells described herein, the cell co-expresses a marker protein for the particular chimeric antigen protein to be expressed. In some alternatives of the cells provided herein, the chimeric antigen receptor is co-expressed with a specific marker protein. In some alternatives of the cells provided herein, the cell comprises a nucleic acid encoding a chimeric antigen receptor. Markers may include selectable marker sequences that confer traits for artificial selection, such as vectors or genes introduced into cells. Selectable marker sequence or marker sequences may be screenable markers that allow a researcher to differentiate between desired and unwanted cells or enrich a particular cell type. In some alternatives, a vector is provided, wherein the vector encodes a chimeric antigen receptor comprising a marker sequence, wherein the marker sequence encodes a cell surface selectable marker. In the alternatives described herein, provided fusion proteins can comprise marker sequences that can be selected in experiments such as flow cytometry. In some alternatives, the marker is the protein Her2tG or EGFRt.

Methotrexate (MTX) may include, for example, antimetabolite and antifolate drugs, but is not limited thereto. In some aspects, it works by inhibiting the metabolism of folic acid. In some alternatives, methods of generating engineered multiplex T cells for adoptive T cell immunotherapy are provided. In the broadest sense, the method may comprise providing any alternative gene transfer polynucleotide described herein, selecting a cell comprising the gene transfer polynucleotide, wherein the selection comprises adding a selection reagent. Include. In some alternatives described herein, the reagent of choice comprises an agent for selection. In some alternatives, the reagent of choice is MTX.

As used herein, "dihydrofolate reductase" or DHFR, as described herein, has its ordinary and general meaning as read in light of the specification, for example tetrahydropol as used in 1-carbon transfer chemistry. As an electron donor that can be converted into a kind of rate cofactor, an enzyme that reduces dihydrofolic acid to tetrahydrofolic acid using NADPH may be included, but is not limited thereto. In some alternatives described herein, gene transfer polynucleotides are provided. In some alternatives, the gene transfer polynucleotide comprises at least one selectable marker cassette encoding a double mutant of dihydrofolate reductase (DHFRdm).

In some embodiments, provided constructs and sequences are modified or optimized, such as by codon optimization, which, for example, changes the codon to a codon known to increase the maximum protein expression efficiency in the desired cell, preferably in human cells. The design process may include, but is not limited thereto. In some alternatives, codon optimization is described, wherein codon optimization can be performed using algorithms known to those of skill in the art to generate synthetic gene transcripts optimized for high protein yield. Programs containing algorithms for codon optimization are known to those skilled in the art. The program may include, for example, OptimumGene™, GeneGPS® algorithms. In addition, synthetic codon optimized sequences can be obtained commercially, for example from Integrated DNA Technologies and other commercially available DNA sequencing services. In some alternatives, a nucleic acid is described wherein the gene of the nucleic acid for the complete gene transcript is codon optimized for expression in humans. In some alternatives, the gene is optimized to have a codon specifically selected for maximum protein expression in human cells, which can increase the concentration of the protein or CAR of the T cell.

Codon optimization can also be performed to reduce the occurrence of secondary structures in polynucleotides. In some alternatives, codon optimization can also be performed to reduce the total GC/AT ratio. Strict codon optimization can also lead to undesirable secondary structures or undesirable GC content leading to secondary structures. As such, the secondary structure affects the transfer efficiency. Programs such as GeneOptimizer can be used to optimize the use of codons and then to avoid secondary structures and to optimize the GC content. These additional programs can be used for further optimization and troubleshooting after initial codon optimization to limit the secondary structures that can occur after the first round of optimization. Alternative programs for optimization are known to those skilled in the art. In some alternatives, the nucleic acid comprises a sequence that is codon optimized for expression and/or secondary structure removal in humans and/or reducing the total GC/AT ratio. In some alternatives, the sequence is optimized for avoiding secondary structures. In some alternatives, the sequence is optimized to reduce the total GC/AT ratio.

As used herein, "vector", "expression vector" or "construct" has its ordinary and general meaning when read in light of the specification, for example having regulatory elements to provide for expression of heterologous nucleic acids in cells. A nucleic acid used to introduce a heterologous nucleic acid into a cell may be included, but is not limited thereto. Vectors include, but are not limited to, plasmid, minicircle, yeast and viral genomes. In some alternatives, the vector is a plasmid, minicircle or viral genome. In some alternatives, the vector is a viral vector. In some alternatives, the viral vector is a lentivirus. In some alternatives, the vector is a lentiviral vector. In some alternatives, the vector is a foaming viral vector, adenovirus vector, retroviral vector or lentiviral vector.

In some embodiments, the T cells or T lymphocytes may comprise T cells from any mammal, preferably a primate, species, including monkeys, dogs and humans. In some alternatives, the T cells are allogeneic (same species but from a different donor) to the recipient subject that will receive or will receive the cells, such as in the form of a therapeutic composition; In some alternatives, T cells are autologous (donor and recipient are the same); In some alternatives, the T cells are syngeneic (donors and recipients are different but identical twins).

As used herein, “ribosomal skip sequence” as described herein refers to a sequence that causes a ribosome to “skip” the ribosome skip sequence during translation and translate the region after the ribosome skip sequence without the formation of a peptide bond. For example, several viruses have ribosomal skip sequences that allow sequential translation of several proteins from a single nucleic acid without having proteins linked through peptide bonds. As described herein, this is a “linker” sequence. In some alternatives of the nucleic acids provided herein, the nucleic acid comprises a ribosome skip sequence between the sequence for the chimeric antigen receptor and the sequence of the marker protein, such that the protein is not linked by peptide bonds and is co-expressed. In some alternatives, the ribosome skip sequence is a P2A, T2A, E2A or F2A sequence. In some alternatives, the ribosome skip sequence is a T2A sequence.

Mature T cells express the surface protein CD4 and are referred to as CD4+ T cells. CD4+ T cells are generally treated as having a predefined role as helper T cells within the immune system. For example, when an antigen-presenting cell expresses an antigen in MHC class II, the CD4+ cells will help that cell through a combination of cell-to-cell interactions (eg, CD40 and CD40L) and through cytokines. Nevertheless, there are rare exceptions; For example, a subgroup of regulatory T cells, natural killer cells, and cytotoxic T cells express CD4. None of the latter CD4+ expressing T cell groups are considered T helper cells.

As used herein, “central memory” T cells (or “T _CM ”) have their mediocre and general meaning when read from the specification, for example expressing CD62L or CCR-7 and CD45RO on their surface and Antigen empirical CTLs that do not express CD45RA or have reduced expression of CD45RA compared to naive cells may be included, but are not limited thereto. In some alternatives, central memory cells are positive for the expression of CD62L, CCR7, CD28, CD127, CD45RO and/or CD95 and have reduced expression of CD54RA compared to naive cells.

As used herein, “effector” “T _E ” T cells have their mediocre and general meaning when read in light of the specification, for example not expressing CD62L, CCR7 or CD28 compared to central memory or naive T cells. Or antigen-experienced cytotoxic T lymphocyte cells that are not limited to or have reduced expression thereof or that are positive for granzyme B and/or perforin.

As used herein, a “pharmaceutical excipient” or pharmaceutical vehicle has its ordinary and general meaning when read in light of the specification, for example as a pharmaceutically active agent for treatment or therapy or as a solvent in which T cells are formulated and/or administered. It may include a carrier or an inert medium used, but is not limited thereto. Vehicles may include polymeric micelles, liposomes, lipoprotein-based carriers, nano-particle carriers, dendrimers, and/or other vehicles for T cells known to those of skill in the art. An ideal vehicle or excipient may be non-toxic, biocompatible, non-immunogenic, biodegradable, and may avoid recognition by the host's defense mechanisms.

As used herein, "T cell precursor" has its mediocre and general meaning when read from the specification, for example a lymphocytic precursor that can migrate to the thymus and become a T cell precursor that does not express T cell receptors. It may include cells, but is not limited thereto. All T cells are derived from hematopoietic stem cells in the bone marrow. Hematopoietic progenitors (lymphoid progenitor cells) from hematopoietic stem cells form a population in the thymus and expand by cell division to produce a large population of immature thymocytes. First thymocytes does not express CD8 Fig without expression also CD4, bi-cell is classified as a voice (CD4 ^{- -} CD8). As they progress through their development, they become double-positive thymocytes (CD4 ⁺ CD8 ⁺ ), and finally mature into single-positive (CD4 ⁺ CD8 ^- or CD4 ^- CD8 ⁺ ) thymocytes, then from the thymus It is released into peripheral tissues.

Glioblastoma polymorphic (GBM) is generally considered to be the most aggressive cancer starting in the brain. The initial signs and symptoms of glioblastoma are generally non-specific. This can include headaches, personality changes, nausea, and stroke-like symptoms.

As used herein, the "leader sequence", also known as the "5' untranslated region (5' UTR), is located upstream from the initiation codon and is a region of the mRNA that is important for regulating the translation of the mRNA transcript. In some alternatives of the method of making genetically modified T cells with chimeric antigen receptor (CAR), the vector encoding the chimeric antigen receptor comprises a sequence encoding a leader sequence.

The ligand is as described in the alternatives herein. “Ligand” has its ordinary and general meaning when read in light of the specification, and may include, for example, a material capable of forming a complex with a biomolecule, but is not limited thereto. By way of example, and not limitation, a ligand can include a substrate, protein, small molecule, inhibitor, activator, nucleic acid or neurotransmitter. Bonding can occur through intermolecular forces such as ionic bonds, hydrogen bonds or van der Waals interactions. Ligands that bind to receptor proteins can alter the three-dimensional structure and determine their functional state. The binding strength of a ligand is referred to as binding affinity and can be determined by direct interaction and solvent effects. Ligands can be linked by “ligand binding domains”. For example, a ligand binding domain can refer to a conserved sequence of structures capable of binding to a specific epitope or specific ligand on a protein. The ligand binding domain or ligand binding moiety may include an antibody or binding fragment or scFv thereof, a receptor ligand or mutant thereof, a peptide, and/or a polypeptide affinity molecule or binding partner. Without limitation, the ligand binding domain can be a specific protein domain or epitope on a protein specific for a ligand or ligands.

Interleukin 13 receptor subunit alpha-2 (IL-13Rα2) is as described in the alternatives herein. "Interleukin-13 receptor subunit alpha-2 (IL-13Rα2)", also known as CD213A2 (cluster of differentiation 213A2), has its mediocre and general meaning when read from the specification, for example in the IL-13RA2 gene in humans. It may include a membrane-binding protein encoded by, but is not limited thereto. IL-13Rα2 is closely related to IL-13Rα1, a subunit of the interleukin-13 receptor complex. IL-13Rα2 generally binds to IL-13 with high affinity, but lacks any significant cytoplasmic domain and does not appear to function as a signal mediator. However, it is possible to modulate the effects of both IL-13 and IL-4 despite the fact that they cannot bind directly to the latter. It is also reported to play a role in the internalization of IL-13.

In some alternatives, the peptide spacer is 15 or fewer amino acids, but 1 or 2 or more amino acids. In some embodiments, the spacer is a polypeptide chain. In some embodiments, the spacer is a polypeptide chain. In some aspects, the polypeptide chain is, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223 , 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239 or 240 amino acids in length, or by any two of the aforementioned lengths. It may be a range of lengths within a defined range. The spacer may contain any 20 amino acids, e.g., in any order to produce a polypeptide chain of the desired length in the chimeric antigen receptor, which is the amino acids arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine. , Asparagine, glutamine, cysteine, glycine, proline, alanine, valine, isoleucine, methionine, phenylalanine, tyrosine and/or tryptophan. The spacer sequence may be a linker between the scFv (or ligand binding domain) and the transmembrane domain of the chimeric antigen receptor. In some alternatives to the chimeric antigen receptor, the chimeric antigen receptor further comprises a sequence encoding a spacer. In some alternatives, the spacers are 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, Defined by the length of 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239 or 240 amino acids, or any two of the aforementioned lengths Includes sequences having a length within the range. In some alternatives, the spacer is between the scFv and the transmembrane region of the chimeric antigen receptor. In some alternatives, the spacer is between the ligand binding domain of the chimeric antigen receptor and the transmembrane region of the chimeric antigen receptor.

Spacers can also be customized, selected or optimized to the desired length to improve binding of the scFv domain to target cells, which can increase cytotoxic efficacy. In some alternatives, the linker or spacer between the scFv domain or ligand binding domain and the transmembrane is 25 to 55 amino acids long (e.g., at least 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55 amino acids or equivalent to or as mentioned above It may be a length within a range defined by any two of the specified lengths).

Some embodiments include a polypeptide sequence or a conservative variant thereof, such as a conservative substitution in a polypeptide sequence. In some embodiments, “conservative amino acid substitution” refers to an amino acid substitution that replaces a functionally-equivalent amino acid. Conservative amino acid changes result in silent changes in the amino acid sequence of the resulting peptide. For example, one or more amino acids of similar polarity act as functional equivalents resulting in silent alterations within the amino acid sequence of the peptide. Substitutions that are charge neutral and replace a moiety with a smaller moiety can also be considered a "conservative substitution" even if the moiety is in a different group (eg, replacing phenylalanine with a smaller isoleucine). Families of amino acid residues with similar side chains have been defined in the art. Conservative amino acid substitutions of several families are shown in Table 1.

<Table 1>

High levels of interleukin 13 receptor alpha 2 (IL-13RΑ2) are found in a number of cancer cells including pancreatic, breast and ovarian cancer or malignant glioma such as glioblastoma. IL-13RΑ2 has also been shown to be overexpressed in the majority of human patients with high-grade astrocytomas (see PLoS One. 2013 Oct 16; 8(10):e77719); the full text of which is expressly incorporated herein by reference. Included). In addition, previous studies have shown that decreasing the amount of IL13RA2 expression in cancer cells significantly slows tumor growth in the model (Breast Cancer Research, 2015; 17 (1); the entirety of which is expressly incorporated herein by reference). . A few types of normal tissues are considered to express only low levels of IL-13-RA2.

For glioblastoma polymorphic (GBM), high expression of IL13Rα2 may be a prognostic marker of tumor progression and poor patient survival. Chimeric antigen receptors (CARs) that selectively target IL13Rα2, and cells and therapies containing or using the same are provided. A provided CAR generally comprises an extracellular domain linked to a transmembrane region and an intracellular signaling region comprising one or more signaling domains, generally comprising primary and costimulatory signaling domains.

The extracellular domain comprises an antigen-binding motif, which in some aspects is or comprises an antigen binding antibody fragment such as scFv. In some aspects, the binding domain, such as scFv, is derived from a human interleukin-13Rα2-specific antibody. In some embodiments, the CAR comprises a spacer region that connects the extracellular binding domain to the transmembrane domain. In some aspects, one or more properties of the spacer optimize the characteristics of the CAR, such as CAR function, by providing flexibility and/or by providing an appropriate distance between the epitope and contact of the binding region and the cell membrane(s). Designed to do. In some aspects, the spacer contains up to 20 amino acids in length, such as up to 15 amino acids in length, up to 14 amino acids in length, up to 13 amino acids in length, or up to 12 amino acids in length. . In some aspects, it contains 12 or 15 or about 12 or 15 amino acids in length. In some aspects, the spacer comprises all or part of an immunoglobulin hinge region (such as a human IgG hinge region, such as a human IgG4 hinge region), or a modified version thereof.

In some alternatives, the spacer comprises a hinge region of a human antibody. In some alternatives of the method, the spacer comprises an IgG4 hinge. In some alternatives, the IgG4 hinge region is a modified IgG4 hinge. A “modified IgG4 hinge” as described herein refers to at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity or as mentioned above. It may refer to a hinge region that may have sequence identity within a range defined by any two of the percentages, and the hinge region amino acid sequence is SEQ ID NO:01, SEQ ID NO:02, SEQ ID NO:03, As described in SEQ ID NO:04, SEQ ID NO:05, SEQ ID NO:06, SEQ ID NO:07 or SEQ ID NO:08.

In some alternatives, the CAR comprises an S spacer, an M spacer, or an L spacer. The S spacer comprises the sequence set forth in SEQ ID NO:09. The M spacer comprises the sequence set forth in SEQ ID NO:10. The L spacer includes the sequence set forth in SEQ ID NO:11.

In some embodiments, the transmembrane portion is or comprises a CD28 transmembrane domain (CD28tm), such as a human CD28 transmembrane domain. CD28tm is encoded by the sequence set forth in SEQ ID NO:12. CD28tm includes the amino acids set forth in SEQ ID NO:13. In some embodiments, the transmembrane domain comprises one or more costimulatory domains, such as a costimulatory domain derived from an intracellular segment of human 4-1BB (CD137) and a signaling domain of CD3-zeta (CD3 ζ), such as human CD3ζ. It is connected to the containing intracellular signaling region. In some alternatives, the CAR design is based on different combinations of two scFv molecules targeting different extracellular epitopes of IL13Rα2, with different VH-VL orientations and three possible length CAR spacers. The remaining CAR backbone, transmembrane and costimulatory domains are shared between CARs. In a further alternative, a truncated version of the epidermal growth factor receptor (EGFRt) that is co-expressed with the CAR on the T cell surface is also included. A further alternative may include a dihydrofolate reductase double mutant (DHFRdm) transgene that allows for methotrexate selection of T-cell products. As described herein, both CD8 and CD4 IL13Rα2 CAR-T cells exhibit potent in vitro tumor cytotoxicity and specific efficacy in vivo against glioblastoma tumor models.

Some alternatives include CARs containing scFvs comprising the VL and VH domains of an antibody referred to as IL13Rα2 Ab01 in the VL-linker-VH orientation. In some aspects, such CARs further comprise specific combination(s) of spacers, transmembrane and/or signaling domains. For example, in some aspects, such a CAR consists of or consists essentially of 15 or fewer or 12 or fewer amino acids and/or an immunoglobulin hinge region or a modified variant thereof, such as the hinge region of IgG4 or a modified thereof. Spacers containing, consisting of, or consisting essentially of the variant. In some aspects, the CAR domain further comprises a costimulatory and primary signaling domain such as 41BB and CD3zeta signaling domain. In some aspects, preferred alternatives are alternative CARs, such as those containing different binding domains (e.g., having different VH and/or VL regions and/or VH and VL regions in different orientations) and/or the same By inducing increased cytotoxicity and cytokine secretion, such as in vitro compared to those containing one or more differences in the other region(s) of the CAR, which contain binding domains but have, for example, alternative spacers, such as spacers with increased length, and /Or it may be advantageous in their ability to enhance T cell effector function by improving function in vivo, such as in the context of human glioblastoma. In some aspects, embodiments provided herein include a subject carrying an IL-13Ra2-expressing tumor in a mouse model comprising a mouse carrying an intracranial-transplanted human GBM, such as treated with a single injection of IL13Rα2 targeted CART-cells. It is based on the observations herein showing the ability to control tumor growth and/or increase median survival.

Specific nucleic acid

Some embodiments of the methods and compositions provided herein include nucleic acids encoding chimeric antigen receptors, the chimeric antigen receptors comprising: a) binding to and/or interacting with the IL-13 alpha 2 (IL13Rα2) receptor. And/or a ligand binding domain comprising CDR3 of the VH region of SEQ ID NO:18, and optionally CDR1 and CDR2, and CDR3 of the VL region of SEQ ID NO:19, and optionally CDR1 and CDR2, b) ligand binding. A polypeptide spacer between the domain and the transmembrane domain; c) transmembrane domain; And d) an intracellular signaling region. In some alternatives, the ligand binding domain is an antibody fragment, such as an antigen-binding fragment. In some alternatives, the ligand binding domain is a single chain variable fragment (scFv). In some alternatives, the spacer is 15 or fewer amino acids, but 1 or 2 or more amino acids. In some alternatives, the spacer comprises the amino acid sequence of X ₁ PPX ₂ P. In some alternatives, the spacer region comprises a portion of the hinge region of a human antibody or a modified variant thereof. In some alternatives, the intracellular signaling region is the signaling domain of CD27, CD28, 4-1BB, OX-40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, NKG2C and B7-H3 and And a primary and costimulatory signaling domain optionally comprising all or part of CD3 zeta in combination with a costimulatory domain selected from the group consisting of combinations thereof. In some alternatives, the intracellular signaling region comprises a signaling portion of CD3 zeta and a signaling portion of 4-1BB. In some alternatives, the transmembrane domain comprises a CD28 transmembrane domain (CD28tm). In some alternatives, the spacer comprises, consists of, or consists essentially of an IgG4 hinge spacer (S). In some alternatives, the spacer comprises, consists of, or consists essentially of an IgG4 hinge-CH3 spacer (M). In some alternatives, the spacer comprises, consists of, or consists essentially of an IgG4 hinge-CH2 (L234D, N297A)-CHE spacer (L). In some alternatives, the scFv includes a VL-VH orientation and spacer S. In some alternatives, the scFv comprises a VH-VL orientation and a spacer, wherein the spacer is a spacer M or a spacer L. In some alternatives, the single chain variable fragment (scFv) comprises the sequence set forth in SEQ ID NO:61 or SEQ ID NO:62. In some alternatives, the single chain variable fragment (scFv) comprises the sequence set forth in SEQ ID NO:63 or SEQ ID NO:64. In some alternatives, the nucleic acid further comprises a nucleic acid encoding a marker sequence. In some alternatives, the marker sequence is a truncated receptor, optionally EGFRt, HER2t or CD19t. In some alternatives, the nucleic acid further comprises a dihydrofolate reductase transgene configured for methotrexate selection. In some alternatives, the dihydrofolate reductase transgene is a dihydrofolate reductase double mutant (DHFRdm). In some alternatives, the first, second, third and/or fourth polynucleotides are codon optimized to reduce the total GC/AT ratio of the nucleic acid. In some alternatives, the first, second, third and/or fourth polynucleotide is codon optimized for expression in humans. In some alternatives, the dihydrofolate reductase double mutant comprises amino acid mutations of L22F and F31S.

Some embodiments of the methods and compositions provided herein include an expression vector comprising the nucleic acid of any of the alternatives herein. In some alternatives, the vector is a viral vector. In some alternatives, the vector is a lentiviral or adenovirus vector.

Specific chimeric receptors

Some embodiments of the methods and compositions provided herein include a nucleic acid of any of the alternatives herein or a chimeric receptor polypeptide encoded by a vector of any of the alternatives herein.

Specific host cell

Some embodiments of the methods and compositions provided herein include host cells comprising a nucleic acid of any of the alternatives herein or an expression vector of any of the alternatives herein, or expressing a chimeric receptor of any of the alternatives herein. In some embodiments, the host cell comprises a genetically modified cell. In some alternatives, the chimeric antigen receptor is encoded by a nucleic acid of any of the alternatives herein or a vector of any of the alternatives herein. In some alternatives, an expression vector comprising the nucleic acid of any of the alternatives herein is provided. In some alternatives, the host cell is a CD8+ T cytotoxic lymphocyte cell selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, and bulk CD8+ T cells. In some alternatives, the CD8+ cytotoxic T lymphocyte cells are central memory T cells, wherein the central memory T cells are positive for CD45RO+, CD62L+ and CD8+. In some alternatives, the host cell is a CD4+ T helper lymphocyte cell selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells. In some alternatives, the CD4+ helper lymphocyte cells are naive CD4+ T cells, wherein the naive CD4+ T cells are positive for CD45RA+, CD62L+ and CD4+ and negative for CD45RO. In some alternatives, the host cell is a precursor T cell. In some alternatives, the host cell is a hematopoietic stem cell.

Some embodiments of the methods and compositions provided herein include a host cell of any of the alternatives herein or a composition of any of the alternatives herein for use in the treatment or inhibition of cancer or solid tumors expressing the IL-13α2 receptor. do. The composition comprises the host cell of any of the alternatives herein and a pharmaceutical excipient. In some alternatives, the cancer is a glioblastoma tumor. In some alternatives, the cancer is glioblastoma polymorphic (GBM). In some alternatives, the cancer is an IL13Rα-positive malignant tumor. In some alternatives, the cancer is brain cancer or brain tumor.

Specific composition

Some embodiments of the methods and compositions provided herein include compositions comprising a host cell of any one of the alternatives herein and a pharmaceutically acceptable excipient.

Host cell specific preparation method

Some embodiments of the methods and compositions provided herein include a method of making a host cell of any one of the alternatives herein, comprising: a) expression of any one of the alternatives herein or any one of the alternatives herein. Introducing the vector into the lymphocyte; And b) culturing the lymphocytes in the presence of an anti-CD3 and/or anti CD28 antibody and at least one homeostatic cytokine until the cells expand; And c) selecting a lymphocyte as a selection reagent; Wherein the selection reagent is configured to selectively enrich cells transduced with a nucleic acid or vector. In some alternatives, the reagent of choice is methotrexate. In some alternatives, the lymphocytes have the CD45RA-, CD45RO+ and CD62L+ phenotypes. In some alternatives, the lymphocyte is CD8+ or CD4+. In some alternatives, the cytokine is IL-15, Il-7 and/or Il-21. In some alternatives, the method further comprises introducing a second nucleic acid into the host cell, the second nucleic acid encoding a marker protein. In some alternatives, the marker protein is EGFRt.

Specific therapy method

Some embodiments of the methods and compositions provided herein are methods of performing cellular immunotherapy in a subject having a cancer or tumor comprising administering to the subject a host cell of any one of the alternatives herein or a composition of the alternative herein. Includes. The composition comprises the host cell of any one of the alternatives herein and a pharmaceutically acceptable excipient. In some alternatives, the cancer is a glioblastoma tumor. In some alternatives, the cancer is glioblastoma polymorphic (GBM). In some alternatives, the cancer is an IL13Rα-positive malignant tumor. In some alternatives, the cancer is brain cancer. In some alternatives, the subject is selected to receive a combination therapy. In some alternatives, combination therapy includes administering a chemotherapy drug. In some alternatives, the combination therapy includes administering radiation therapy. In some alternatives, chemotherapy drugs are electrochemotherapy, alkylating agents, antimetabolites (e.g., 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine (geloda ®), cladribine, cloparabine, cytarabine (Ara-C®), phloxuridine, fludarabine, gemcitabine (Gemza®), hydroxyurea, methotrexate, pemetrexide (Alymta®), pento Statins, and thioguanine), anti-tumor antibiotics, topoisomerase inhibitors, mitosis inhibitors, corticosteroids, DNA intercalating agents, or checkpoint inhibitors (checkpoint kinases CHK1, CHK2). In some alternatives, the cancer is a glioma.

Development of specific IL13Ra2 CAR

A schematic diagram of a nucleic acid encoding the structure of several generated IL-13Rα2 CARs is shown in FIG. 1, each of which has one of the various binding domains, one of the various spacer regions, with a transmembrane domain and an intracellular signaling domain(s). Have it together The nucleic acid sequence of each of these exemplary IL-13R13Rα2 CARs comprises a leader sequence, which can be used, for example, to allow translation of the mRNA transcript encoding the CAR. Each of the exemplary CARs is also an IL-13Rα2 scFv, specifically an anti-IL13Rα2 scFv having the VH and VL domains set forth in SEQ ID NOs: 14 and 16, respectively, or the VH and VL domains set forth in SEQ ID NOs: 15 and 17, respectively. Included. Each of the generated and tested CARs was generated with 4 different scFv binding domains. The four binding domains individually contained each anti-IL13Rα2 VH/VL pair in respective VL-VH and VH-VL orientations.

Three different spacers that are individually present in the exemplary CAR are shown in Figure 1 and are located between the scFv and the transmembrane domain. The tested spacers varied in length and were derived from immunoglobulin constant regions. The spacer with the shortest amino acid sequence length was a modified human IgG4 hinge region), and the spacer with the median (medium) length contained a modified human hinge region and further contained a CH3 domain (IgG4-CH3); The longest spacers tested included a modified IgG4 hinge region, a modified CH2 region and a CH3 region (IgG4-CH2 (L235D, N297Q)-CH3 (L)). Each of the resulting exemplary CARs further comprised a transmembrane derived from human CD28 (CD28tm domain) and a signaling domain of human 4-1BB and CD3 zeta (4-1BB domain, CD3 zeta domain).

Each nucleic acid used to encode and express the CAR further included a sequence encoding a T2A skip sequence and a nucleic acid encoding a truncated surrogate marker for CAR expression.

In some alternatives, the ligand binding domain of an embodiment of a provided CAR has or consists of the amino acid sequence of SEQ ID NO: 18, or at least 90, 91, 92, 93, 94, 95, 96 with SEQ ID NO: 18. , A heavy chain variable region comprising or consisting of a sequence having 97, 98 or 99% identity, or comprising a CDR1, CDR2 and/or CDR3 of a VH region having the sequence of SEQ ID NO: 18, typically at least CDR3. Include.

In some alternatives, the ligand binding domain of embodiments of a provided CAR has or consists of the amino acid sequence of SEQ ID NO: 19, or at least 90, 91, 92, 93, 94, 95, 96 with SEQ ID NO: 19 , A light chain variable region comprising CDR1, CDR2 and CDR3 of a VL region having a sequence having 97, 98 or 99% identity, or consisting of, or having the sequence of SEQ ID NO:19. Sequences are described in WO2014072888, which is incorporated herein by reference in its entirety.

In some alternatives, the ligand binding region that specifically binds human IL-13-Ra2 comprises (a) a heavy chain CDR1 comprising SEQ ID NO:20; (b) a heavy chain CDR2 comprising SEQ ID NO:21; (c) comprises a heavy chain CDR3 comprising SEQ ID NO:22 and/or; (d) a light chain CDR1 comprising SEQ ID NO:23; (e) a light chain CDR2 comprising SEQ ID NO:24; And/or (f) a light chain CDR3 comprising SEQ ID NO:25.

In some alternatives, the ligand binding region comprises a heavy chain variable region amino acid sequence of SEQ ID NO:18 and a light chain variable region amino acid sequence of SEQ ID NO:19.

In some alternatives, the ligand binding domain comprises a heavy chain CDR2 (CDRH2) comprising SEQ ID NO:28; (c) a heavy chain CDR3 (CDRH3) comprising SEQ ID NO:29; (d) a light chain CDR1 comprising SEQ ID NO:30; (e) a light chain CDR2 comprising SEQ ID NO:31; And/or (f) a light chain CDR3 comprising SEQ ID NO:32.

In some alternatives, the ligand binding domain comprises a heavy chain CDR1 (CDR-H1) comprising a sequence set forth in SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, or SEQ ID NO:36. In some alternatives, the CDR-H2 comprises a sequence set forth in SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, or SEQ ID NO:40. In some alternatives, CDRH3 comprises the sequence set forth in SEQ ID NO:41.

In some embodiments, the CAR comprises a spacer interposed between the binding domain and the transmembrane domain. In some alternatives, the spacer comprises, consists of, or consists essentially of at least a portion of an immunoglobulin constant region, such as all or part of an immunoglobulin hinge region, such as an IgG4 hinge region, or a functional variant thereof. In some embodiments, the immunoglobulin constant and/or hinge region is a region of a human IgG, such as IgG4 or IgG1, or a variant thereof. The spacer can be of a length that provides increased reactivity of the cell after antigen binding compared to in the absence of the spacer. Exemplary spacers include those described in International Patent Application Publication No. WO2014031687, which is expressly incorporated herein by reference in its entirety. In some examples, the spacer is 12 or about 12 amino acids in length, 12 or less amino acids in length, 15 or about 15 amino acids in length, or 15 or less or about 15 amino acids in length. .

Exemplary spacers are at least 10 to 229 amino acids, 10 to 200 amino acids, 10 to 175 amino acids, 10 to 150 amino acids, 10 to 125 amino acids, 10 to 100 amino acids, 10 to 75 amino acids, 10 to 50 amino acids. Have amino acids, 10 to 40 amino acids, 10 to 30 amino acids, 10 to 20 amino acids, or 10 to 15 amino acids, and include any integer between the endpoints of any of the enumerated ranges. In some embodiments, the spacer region has 12 or fewer but non-zero amino acids, 119 or fewer but non-zero amino acids, or 229 or fewer but non-zero amino acids. In some embodiments, the spacer is less than 250 but not zero amino acids in length, less than 200 but not zero amino acids in length, less than 150 but not zero amino acids in length, less than 100 amino acids in length but zero. Non-dog amino acids, less than 75 amino acids in length but not zero, less than 50 amino acids in length but not zero amino acids, less than 25 amino acids in length but not zero amino acids, less than 20 amino acids in length but zero Not amino acids, less than 15 amino acids in length but not zero amino acids, less than 12 amino acids in length but not zero amino acids, or less than 10 amino acids in length but not zero amino acids. In some embodiments, the spacer is 10 to 250 amino acids in length, 10 to 150 amino acids in length, 10 to 100 amino acids in length, 10 to 50 amino acids in length, 10 to 25 amino acids in length, 10 To 15 amino acids, 15 to 250 amino acids in length, 15 to 150 amino acids in length, 15 to 100 amino acids in length, 15 to 50 amino acids in length, 15 to 25 amino acids in length, 25 to 250 amino acids in length Amino acids, 25 to 100 amino acids in length, 25 to 50 amino acids in length, 50 to 250 amino acids in length, 50 to 150 amino acids in length, 50 to 100 amino acids in length, 100 to 250 amino acids in length , 100 to 150 amino acids in length, or 150 to 250 amino acids in length. Exemplary spacers include an IgG4 hinge alone, an IgG4 hinge linked to the CH2 and CH3 domains, or an IgG4 hinge linked to the CH3 domain. Exemplary spacers are described in Hudecek et al. Clin. Cancer Res., 19:3153 (2013)], including, but not limited to, those described in International Patent Application Publication No. WO2014031687, US Patent No. 8,822,647 or Published Application No. US2014/0271635, which are incorporated herein by reference in their entirety. Included explicitly.

In some aspects, the spacer of the CAR consists of or comprises an IgG4 hinge, an IgG4 hinge-CH3 or an IgG4 hinge-CH2(L235D, N297Q)-CH3.

In some alternatives, the ligand binding domain comprises (a) a heavy chain variable region comprising CDR1, CDR2 and CDR3 of the VH sequence of SEQ ID NO:18; And (b) a light chain variable region comprising CDR1, CDR2 and CDR3 of the VL sequence of SEQ ID NO: 19. The sequence can be found in WO2014/072888, which is expressly incorporated herein by reference in its entirety.

In some alternatives, the orientation of the chain is VL-linker-VH. In some aspects, the orientation is VH-linker VL.

In some alternatives, the ligand binding region comprises a heavy chain variable region comprising CDR1, CDR2 and CDR3 of the VH sequence shown in SEQ ID NO:18; It includes a light chain variable region comprising CDR1, CDR2 and CDR3 of the VL sequence shown in SEQ ID NO: 19. In some alternatives, a spacer is provided, wherein the spacer comprises an immunoglobulin constant region, such as an Fc region comprising at least one pair of amino acid substitutions selected from the group consisting of the amino acid sequence of SEQ ID NO:42 and SEQ ID NO:43. Including all or part of; Or L443C (SEQ ID NO:44), Q347C (SEQ ID NO:45), kK183C (SEQ ID NO:46), SEQ ID NO:47, L443C/kK183C (SEQ ID NO:44 and SEQ ID NO:46) , At least one engineered light chain constant region selected from the group consisting of Q347C/kA111 C (SEQ ID NO:45 and SEQ ID NO:47), and Q347C/kK183C (SEQ ID NO:45 and SEQ ID NO:46), and It contains an engineered Fc region.

In some alternatives, the constant region or portion thereof comprises the sequence set forth in SEQ ID NO:48.

In some alternatives, the Ab02-derived scFv comprises the sequence set forth in SEQ ID NO:49 (LCDR1), SEQ ID NO:50 (LCDR2) and/or SEQ ID NO:51 (LCDR3). In some alternatives, the Ab02 antibody comprises a sequence as set forth in SEQ ID NO:55 (HCDR1), SEQ ID NO:56 (HCDR2) and/or SEQ ID NO:57 (HCDR3).

In some alternatives, the Ab01 antibody comprises the sequence set forth in SEQ ID NO:52 (LCDR1), SEQ ID NO:53 (LCDR2) and/or SEQ ID NO:54 (LCDR3). In some alternatives, the Ab01 antibody comprises a sequence as set forth in SEQ ID NO:58 (HCDR1), SEQ ID NO:59 (HCDR2) and/or SEQ ID NO:60 (HCDR3).

In some embodiments, the CAR comprises or comprises an antigen-binding region comprising an scFv comprising VH and VL domains in a VL-linker-VH orientation. In some embodiments, such as in some aspects of these embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:18, the CDR3 (and, optionally, CDRs 1 and 2) of the VH sequence set forth in SEQ ID NO:18. And/or comprises or consists of an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO:18, or of SEQ ID NO:18 CDR1, CDR2 and/or CDR3 of the VH region having sequence, typically at least CDR3; For example, in some aspects of these embodiments, the VH is a VH derived from Ab01. In some aspects of these embodiments, the VL domain comprises the amino acid sequence of SEQ ID NO: 19, the CDR3 (and, optionally, CDRs 1 and 2) of the VL sequence set forth in SEQ ID NO: 19, and/or the SEQ ID NO: Of a VL region comprising or consisting of an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 19, or having the sequence of SEQ ID NO: 19 CDR1, CDR2 and/or CDR3, typically at least CDR3; For example, in some aspects of these embodiments, the VH is a VH derived from Ab01. In some aspects of any of these embodiments, such as those having such binding domains, the CAR is 50, 40, 30 or 20 or less or about 50, 40, 30 or 20 amino acids or less in length, such as 15 A binding domain containing, consisting of, or consisting essentially of, an immunoglobulin hinge region or a variant thereof, such as an IgG4 hinge or a variant thereof, and/or contains no more than about 15 or no more than 12 or no more than about 12 amino acids, and It includes spacer regions between the transmembrane domains.

In some aspects, the CAR and/or cells expressing it display one or more increased or superior functional activity compared to a reference CAR. In some aspects, the reference CAR has the same binding domain (and optionally otherwise the same) but differs in length and/or composition, such as 50, 40, 30, 20, 15 or 12 or about 50, 40, 30, CARs having a spacer that is one more amino acid than 20, 15 or 12 amino acids, and/or a CAR having the same or similar variable region but with a binding domain having an alternative orientation. In some embodiments, the function is an in vitro assay using cells expressing IL-13Ra2, such as a co-culture assay (e.g., an assay measuring cytokine production or cytolytic activity) or an animal with a tumor that is positive for IL-13Ra2. As observed in an in vivo assay using a model, such as an assay to assess tumor burden reduction or survival following administration of cells expressing CAR, such as T cells.

In an alternative herein, 8 CARs with various combinations of antigen-binding domains and other domains as shown in FIG. 1 were tested in various assays.

Specific sequence

In some alternatives, the Ab01 VH comprises the sequence set forth in SEQ ID NO:14. In some alternatives, the Ab01 VL comprises the sequence set forth in SEQ ID NO:16. In some alternatives, the IL13Ra2 Ab01 VLVH scFv is encoded by the sequence set forth in SEQ ID NO:61. In some alternatives, the IL13Ra2 Ab01 VHVL scFv is encoded by the sequence set forth in SEQ ID NO:62, which is the Ab01-VL sequence, Ab01-VH sequence, ATG start codon, 5'NheI restriction site (GCTAGC) and 3'RsrII restriction. Site (CGGACCG). In some alternatives, the Ab01-VH protein sequence comprises SEQ ID NO:65. In some alternatives, the Ab01 HCDR1 comprises the sequence set forth in SEQ ID NO:66. In some alternatives, the Ab01 HCDR2 comprises the sequence set forth in SEQ ID NO:67. In some alternatives, the Ab01 HCDR3 comprises the sequence set forth in SEQ ID NO:68. In some alternatives, Ab01-VL comprises the sequence set forth in SEQ ID NO:69. In some alternatives, Ab01 LCDR1 comprises the sequence set forth in SEQ ID NO:70. In some alternatives, Ab01 LCDR2 comprises the sequence set forth in SEQ ID NO:71. In some alternatives, Ab01 LCDR3 comprises the sequence set forth in SEQ ID NO:72.

In some alternatives, Ab02 VH comprises the sequence set forth in SEQ ID NO:15. In some alternatives, the Ab02 VL comprises the sequence set forth in SEQ ID NO:17. In some alternatives, the IL13Ra2 Ab02 VLVH scFv is encoded by the sequence set forth in SEQ ID NO:63. In some alternatives, the IL13Ra2 Ab02 VHVL scFv is encoded by the sequence set forth in SEQ ID NO:64, which is the Ab02-VL sequence, Ab02-VH sequence, ATG start codon, 5'NheI restriction site (GCTAGC) and 3'RsrII restriction Site (CGGACCG). In some alternatives, the Ab02-VH protein sequence comprises the sequence set forth in SEQ ID NO:73. In some alternatives, Ab02 HCDR1 comprises the sequence set forth in SEQ ID NO:74. In some alternatives, Ab02 HCDR2 comprises the sequence set forth in SEQ ID NO:75. In some alternatives, Ab02 HCDR3 comprises the sequence set forth in SEQ ID NO:76. In some alternatives, the Ab02-VL protein sequence comprises the sequence set forth in SEQ ID NO:77. In some alternatives, Ab02 LCDR1 comprises the sequence set forth in SEQ ID NO:78. In some alternatives, Ab02 LCDR2 comprises the sequence set forth in SEQ ID NO:79. In some alternatives, Ab02 LCDR3 comprises the sequence set forth in SEQ ID NO:80.

<Example>

Example 1- Construction of IL-13Rα2-targeting CAR

Various IL-13Rα2-targeting CARs were constructed. As shown in Figure 1, IL-13Rα2 specific CARs included: Among the numerous single-chain variable fragments (scFvs) that specifically recognize the extracellular epitope of the IL-13 alpha 2 receptor (IL-13Rα2). one; One of the various spacer domains (denoted as “S”, “M” and “L”); A transmembrane domain derived from human CD28 (CD28tm); A costimulatory domain derived from human 4-1BB; CD3ζ-derived signaling domain; T2A ribosome skip sequence; And a truncated EGFR (EGFRt) transduction marker; And a dihydrofolate reductase double mutant (DHFRdm) transgene, optionally configured for additional T2A ribosomal skip sequence and methotrexate selection.

Examples of antibodies or epitope binding fragments from which single-chain variable fragments (scFv) can be derived are humanized anti-IL-13-Ra2 IgG1 antibody hu08 (Creative Biolabs, NY), and anti-IL13RA2 treatment. Antibody (hu07 v1.1) (Creative Biolabs, NY).

The spacer domain was linked to the scFv in each CAR, and the extracellular binding element was linked to the transmembrane domain. Each CAR has three different spacers (S, M, 11) each having a different length amino acid sequence (spacer S, SEQ ID NO:9; spacer M, SEQ ID NO:10; or spacer L, SEQ ID NO:11). Or L). Different scFv configurations are used individually in various IL-13Rα2 CARs comprising VH and VL domains derived from one of two different anti-IL-13Ra2-directed antibodies ('Hu08', Ab01; and'Hu07',Ab02) Became. The scFv portion of each CAR is present in the VL-VH or VH-VL orientation, linked by a flexible peptide linker, a VH region having a sequence as set forth in SEQ ID NO:14 or SEQ ID NO:15 and SEQ ID NO: :16 or a VL region having the sequence set forth in SEQ ID NO:17.

Example 2-Functional comparison of CARs derived from different antibodies

Functional comparisons of CARs comprising scFv binding domains derived from different antibodies in different orientations were performed. Comparison included surface expression analysis of EGFRt and cytokine release assay. In this study, each CAR is a "S" spacer (having the amino acid sequence of SEQ ID NO:9, a modified immunoglobulin hinge region), as well as the transmembrane and costimulatory and primary signaling described and shown in Figure 1 The domain was included with one of the various scFv binding regions (VH/VL domains and orientations) shown in FIG. 1. CARs included: IL-13Ra2 Ab01 VLVH spacer S CAR; IL-13Ra2 Ab01 VHVL spacer S CAR; IL-13Ra2 Ab02 VLVH spacer S CAR; And IL-13Ra2 Ab02 VHVL spacer S CAR.

Expression of CAR in cells was examined by determining the expression of EGFRt markers in CD8+ and CD4+ T cells using flow cytometry (FIG. 2A ).

CAR T cells were individually co-cultured with DAOY cells engineered to express IL13Rα2 (human brain/cerebellar cell line), U251T cells (human glioblastoma cell line) and K562 cells (human chronic myelogenous leukemia cells). After 24 hours, the levels of IL-2, IFN-γ and TNF-α were measured in the supernatant using a cytokine assay. Increased cytokine production after incubation with target cells was observed for CD8+ and CD4+ T cells expressing CARs containing binding domains with VH and VL domains from Ab01 antibody in both VH/VL orientations (Figure 2B and Figure 2c).

Example 3-Functional comparison of CARs containing different spacers

Functional comparisons of CARs comprising different spacers of different lengths and scFvs derived from the same antibody (in different orientations) were performed. The four CARs shown in Figure 1 were tested for cell surface expression in CD8+ by gating against the marker sequence, EGFRt (Figure 3A). In addition, CD8+ cells bearing CAR were also tested for their ability to induce specific lysis, as well as their ability to release cytokines. In this study, each exemplary CAR is one of two different binding domains (scFv with VL and VH domains from Ab01 in VL-VH or VH-VL orientation) and various spacers (amino acid sequence of SEQ ID NO:9). "S" having, "M" having the amino acid sequence of SEQ ID NO:10, and "L" having the amino acid sequence of SEQ ID NO:11), as well as the transmembrane and cavity described and shown in FIG. Stimulation and primary signaling domains were included.

Representative results from multi-parameter flow cytometry analysis of T cells derived from healthy donors and transduced with the indicated CAR lentivirus are shown in Figure 3A (top and middle panels), where the histogram quadrants are plotted based on control staining. lost. Surface surrogate marker (EGFRt) expression on CD8+ IL-13Rα2 CAR T-cells was analyzed by flow cytometry (Fig. 3A, lower panel).

IL-13Rα2-targeting CAR expressing CD8 T cells were co-cultured with different IL-13Ra2-expressing target cell lines with variable indicated effector to target ratios (FIG. 3B ). Cytolytic activity was evaluated using a chromium release assay, and cytokine production was evaluated in the supernatant. The chromium release assay took place over a 4 hour co-culture period, but cytokine release was assessed after 24 hours co-culture (2:1 effector-target ratio). As shown in FIG. 3C, co-culture with various CAR-expressing T cells increasingly induced specific lysis with increased effector to target ratio, and cytokine secretion levels. Compared to other CARs evaluated, increased cytolytic activity was observed for cells expressing a CAR with an “S” spacer and containing scFvs with VL-VH orientation. In each evaluated CAR having a binding domain with VL-VH orientation, target-specific cytolytic activity and cytokine production were observed. In addition, it was observed that in CARs with VL-VH orientation, the presence of “S” spacers resulted in superior effects compared to other spacers tested. In contrast, no cytolytic activity was observed in this assay for CARs with VH-VL orientation and "S" spacers (while for each VH-VL CAR with "M" and "L" spacers the activity was Observed).

Regarding cytokine production, as shown in FIG. 3B, T cells with IL-13Ra2 Ab01 VLVH spacer S CAR, T cells with IL-13Ra2 Ab01 VLVH spacer M CAR, and T cells with IL-13Ra2 Ab01 VLVH spacer L CAR Caused higher expression of IFN-gamma. T cells with IL-13Ra2 Ab01 VHVL spacer M CAR, and T cells with IL-13Ra2 Ab01 VHVL spacer L CAR showed IFN-gamma expression. T cells with IL-13Ra2 Ab01 VLVH spacer S CAR, T cells with IL-13Ra2 Ab01 VLVH spacer M CAR, and T cells with IL-13Ra2 Ab01 VLVH spacer L CAR induced higher specific lysis.

Regarding specific lysis, as shown in Figure 3b, at increased concentrations, CD8+ cells carrying IL-13Rα2 Ab01 VLVH spacer S CAR, IL-13Rα2 Ab01 VLVH intermediate CAR, and IL-13Rα2 Ab01 VLVH long CAR are high. It caused a significant amount of specific lysis in the concentration of cells. In addition, these cells induced high production of the cytokine IFN-gamma.

In contrast, cells expressing IL-13Rα2 Ab01 VHVL spacer S CAR, IL-13Rα2 Ab01 VHVL intermediate CAR and IL-13Rα2 Ab01 VHVL long CAR are IL-13Rα2 Ab01 VLVH spacer S CAR, IL-13Rα2 Ab01 VLVH intermediate CAR and IL -13Rα2 Ab01 VLVH had a reduced amount of specific lysis compared to long CAR. No specific lysis was observed in the IL-13Rα2 Ab01 VHVL spacer S CAR. In addition, IL-13Rα2 Ab01 VHVL spacer S CAR expression did not induce IFN-gamma expression, which was seen in IL-13Rα2 Ab01 VHVL intermediate CAR and IL-13Rα2 Ab01 VHVL long CAR.

Example 4- In vivo anti-tumor activity of anti-IL13Rα2 CAR

After administration of cells expressing IL13Rα2-specific CAR containing the binding domain derived from antibody Ab01, VLVH orientation, and spacer "S", the treatment results were evaluated in a mouse glioblastoma model. The CAR used in the experiment was expressed in CD8+ cells. For the experiment, ffLuc+ U87 glioblastoma cells (0.2 x 10 ⁶ cells) were injected intracranially into the forebrain of NSG mice on day 0. On day 7, mice (n=3 per group) were not treated (vehicle control or mock), or different doses of IL-13Rα2 Ab01 VLVH transduced CD8+ CAR T-cells (2 x 10 ⁶ , 1 x 10 ⁶ or 0.5 x 10 ⁶ cells).

4A and 4B are in vivo IL13Rα2-specific CARs containing VH and VL domains derived from Ab01 and “S” spacers in VL-VH orientation with transmembrane and signaling domains as shown in FIG. 1 Results from studies demonstrating anti-tumor activity are shown. Human glioblastoma cells expressing firefly luciferase (ffLuc ⁺ U87 glioblastoma cells) (0.2x10 ⁶ cells) were injected intracranially into the forebrain of NSG mice on day 0. On day 7, mice (n=3 per group) received vehicle control or sham administration, or at different doses of CD8+ cells expressing IL-13Rα2-specific CAR (Ab01 VL-VH) (2 x 10 ⁶ , 1 x 10 ⁶ or 0.5 x 10 ⁶ cells) were administered. Figure 4A shows tumor burden (y) over time (days after tumor inoculation, x-axis) for animals treated with vehicle or sham treatment (left) and indicated decreasing number of CAR+ cells (right; right to left) -Axis) as a measure of total flux (photons/sec), which shows tumor regression after administration of cells expressing CAR. Kaplan-Meier survival curves showed increasingly improved survival for mice treated with increasing doses of T cells expressing anti-IL-13Rα2 CAR (FIG. 4B ). Vehicle control had a 0% survival rate on day 25. However, mice that received 2 x 10 ⁶ CD8+ T-cells expressing IL13Rα2 Ab01 VLVH had ˜50% survival after 90 days.

Example 5- In vivo anti-tumor activity of anti-IL13Rα2 CAR

To evaluate the effect of the IL13Ra2 Ab01 VLVH CAR spacer, an experiment substantially similar to Example 3 was performed using U251T tumor-bearing mice as a glioblastoma model. On day 0, U251T GFP:ffluc tumor cells were injected intracranially, and on day 7, CD8+ CAR T-cells or Cd8+ mock T-cells were injected intratumorally. Mice injected with mock T-cells were used as controls.

All mice treated with IL13Rα2 Ab01 VLVH 2G short CAR T-cells showed complete tumor regression. Only one of the CAR-median mouse groups showed tumor recurrence and required animal euthanasia at day 58 after tumor injection. Three of the U251T tumor-bearing mice treated with intratumoral injection of long spacer IL13Rα2 CAR T-cells showed less therapeutic activity and tumor recurrence.

Taken together, all these data suggest that the extracellular short spacer size is the optimal configuration for IL13Rα2 Ab01 VLVH conferring optimal in vivo anti-tumor efficacy, which supported its clinical development.

Table 2 lists specific amino acid and nucleotide sequences for some embodiments of the methods and compositions provided herein.

<Table 2>

With respect to the use of plural and/or singular terms herein, one of ordinary skill in the art may translate plural to singular and/or singular to plural as appropriate to the context and/or application. For clarity, various singular/plural permutations may be explicitly set forth herein.

One of ordinary skill in the art will generally understand that the terms used herein and in particular in the appended claims (eg, the text of the appended claims) are generally intended as “open” terms (see For example, the term “including” should be interpreted as “including but not limited to”, the term “having” should be interpreted as “having at least”, and the term “includes” Should be interpreted as "including but not limited to"). It will be further understood by those of ordinary skill in the art that where a particular number of introduced claim recitations are intended, such intent will be expressly recited in the claims, and in the absence of such citations that such intent does not exist. For example, to aid understanding, the following appended claims may contain the use of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases is interpreted to mean that the introduction of a claim recitation by the singular form “a” limits any particular claim containing such an introduced claim recitation to embodiments containing only one such citation. (E.g., "one" means "at least one" or "one or more", even if the same claim includes the introductory phrase "one or more" or "at least one" and the singular form, such as "one" Should be interpreted as)); The same applies to the use of the definite article used to introduce claim recitation. In addition, even if a certain number of introduced claim citations are explicitly cited, those skilled in the art will recognize that such citations should be interpreted to mean at least the number recited (e.g., "2 An as-is citation of "four citations" means at least 2 citations, or 2 or more citations). Further, when a rule similar to "at least one of A, B and C, etc." is used, in general, such construction is intended to mean that one of ordinary skill in the art will understand the rule (eg, "A , A system having at least one of B and C" refers to a system having A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B and C together, etc. Including but not limited to). Where rules similar to “at least one of A, B or C, etc.” are used, in general, such construction is intended to mean that one of ordinary skill in the art will understand the rules (eg, “A, B Or a system having at least one of C” includes systems having A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B and C together, etc. But not limited to this). Contemplating the possibility that virtually any conjunctive word and/or phrase suggesting two or more alternative terms, whether in the description, claims, or drawings, will include one, any of the terms, or both terms. Those skilled in the art will further understand that it should be understood. For example, the phrase “A or B” will be understood to include the possibility of “A” or “B” or “A and B”.

In addition, where a feature or aspect of the present disclosure is described with respect to a Markush group, those skilled in the art will appreciate that the disclosure is also described herein with respect to any individual member or subgroup of members of the Markush group. Will recognize.

Any features of the embodiments of the first to eighth aspects are applicable to all aspects and embodiments identified herein. Moreover, any features of the embodiments of the first to eighth aspects are partially or wholly independently combinable with the other embodiments described herein in any way, for example 1, 2 or 3 or more embodiments It may be wholly or partially combinable. In addition, any feature of the embodiments of the first to eighth aspects can be made optional with respect to other aspects or embodiments.

SEQUENCE LISTING <110> Giacomo Tampella Michael C. Jensen <120> IL-13 RECEPTOR ALPHA 2 (IL13RA2) CHIMERIC ANTIGEN RECEPTOR FOR TUMOR SPECIFIC T CELL IMMUNOTHERAPY <130> SCRI.169WO <150> 62/643055 <151> 2018-03-14 <160> 101 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Human IgG1 <400> 1 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 15 <210> 2 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Human IgG2 <400> 2 Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro 1 5 10 <210> 3 <211> 61 <212> PRT <213> Artificial Sequence <220> <223> Human IgG3 <400> 3 Glu Leu Lys Thr Pro Leu Gly Asp Thr His Thr Cys Pro Arg Cys Pro 1 5 10 15 Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu 20 25 30 Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu Pro 35 40 45 Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 50 55 60 <210> 4 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Human IgG4 <400> 4 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro 1 5 10 <210> 5 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Modified Human IgG4 <400> 5 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 1 5 10 <210> 6 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Modified Human IgG4 <400> 6 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 1 5 10 <210> 7 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Modified Human IgG4 <400> 7 Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 1 5 10 <210> 8 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Modified Human IgG4 <400> 8 Glu Val Val Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 1 5 10 <210> 9 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> S spacer <400> 9 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 1 5 10 <210> 10 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> M spacer <400> 10 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gln Pro Arg 1 5 10 15 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 20 25 30 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 35 40 45 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 50 55 60 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 65 70 75 80 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 85 90 95 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 100 105 110 Leu Ser Leu Ser Leu Gly Lys 115 <210> 11 <211> 229 <212> PRT <213> Artificial Sequence <220> <223> L spacer <400> 11 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe 1 5 10 15 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225 <210> 12 <211> 84 <212> DNA <213> Artificial Sequence <220> <223> CD28tm <400> 12 atgttctggg tgctggtggt ggtcggaggc gtgctggcct gctacagcct gctggtcacc 60 gtggccttca tcatcttttg ggtg 84 <210> 13 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> CD28tm <400> 13 Met Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser 1 5 10 15 Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 20 25 <210> 14 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Ab01-VH <400> 14 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Asn 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Thr Val Ser Ser Gly Gly Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Gly Thr Thr Ala Leu Ala Thr Arg Phe Phe Asp Val Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 15 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Ab02-VH <400> 15 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Lys Tyr 20 25 30 Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Lys Trp Ala Gly Gly Ser Thr Asp Tyr Asn Ser Ala Leu Met 50 55 60 Ser Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp His Arg Asp Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 16 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Ab01-VL <400> 16 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Ser Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Ser Ala Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 17 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Ab02-VL <400> 17 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Thr Ala Ser Leu Ser Val Ser Ser Thr 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln Tyr His Arg Ser Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 18 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> VH region <400> 18 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Asn 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Thr Val Ser Ser Gly Gly Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Gly Thr Thr Ala Leu Ala Thr Arg Phe Phe Asp Val Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 19 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL region <400> 19 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Ser Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Ser Ala Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 20 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> heavy chain CDR1 <400> 20 Ser Arg Asn Gly Met Ser 1 5 <210> 21 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> heavy chain CDR2 <400> 21 Thr Val Ser Ser Gly Gly Ser Tyr Ile Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 22 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> heavy chain CDR3 <400> 22 Gln Gly Thr Thr Ala Leu Ala Thr Arg Phe Phe Asp Val 1 5 10 <210> 23 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> light chain CDR1 <400> 23 Lys Ala Ser Gln Asp Val Gly Thr Ala Val Ala 1 5 10 <210> 24 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> light chain CDR2 <400> 24 Ser Ala Ser Tyr Arg Ser Thr 1 5 <210> 25 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> light chain CDR3 <400> 25 Gln His His Tyr Ser Ala Pro Trp Thr 1 5 <210> 26 <400> 26 000 <210> 27 <400> 27 000 <210> 28 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> heavy chain CDR2 <400> 28 Thr Val Ser Ser Gly Gly Ser Tyr Ile Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 29 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> heavy chain CDR3 <400> 29 Gln Gly Thr Thr Ala Leu Ala Thr Arg Phe Phe Asp Val 1 5 10 <210> 30 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> light chain CDR1 <400> 30 Lys Ala Ser Gln Asp Val Gly Thr Ala Val Ala 1 5 10 <210> 31 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> light chain CDR2 <400> 31 Ser Ala Ser Tyr Arg Ser Thr 1 5 <210> 32 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> light chain CDR3 <400> 32 Gln His His Tyr Ser Ala Pro Trp Thr 1 5 <210> 33 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> heavy chain CDR1 <400> 33 Gly Phe Thr Phe Ser Arg Asn 1 5 <210> 34 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> heavy chain CDR1 <400> 34 Gly Phe Thr Phe Ser Arg Asn Gly Met Ser 1 5 10 <210> 35 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> heavy chain CDR1 <400> 35 Arg Asn Gly Met Ser 1 5 <210> 36 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> heavy chain CDR1 <400> 36 Ser Arg Asn Gly Met Ser 1 5 <210> 37 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> heavy chain CDR2 <400> 37 Ser Ser Gly Gly Ser Tyr 1 5 <210> 38 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> heavy chain CDR2 <400> 38 Thr Val Ser Ser Gly Gly Ser Tyr Ile Tyr 1 5 10 <210> 39 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> heavy chain CDR2 <400> 39 Thr Val Ser Ser Gly Gly Ser Tyr Ile Tyr 1 5 10 <210> 40 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> heavy chain CDR2 <400> 40 Thr Val Ser Ser Gly Gly Ser Tyr Ile Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 41 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> heavy chain CDR3 <400> 41 Ala Arg Gln Gly Thr Thr Ala Leu Ala Thr Arg Phe Phe Asp Val 1 5 10 15 <210> 42 <211> 452 <212> PRT <213> Artificial Sequence <220> <223> Fc region <400> 42 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Asn 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Thr Val Ser Ser Gly Gly Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Gly Thr Thr Ala Leu Ala Thr Arg Phe Phe Asp Val Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Cys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Cys 435 440 445 Ser Pro Gly Lys 450 <210> 43 <211> 452 <212> PRT <213> Artificial Sequence <220> <223> Fc region <400> 43 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Asn 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Thr Val Ser Ser Gly Gly Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Gly Thr Thr Ala Leu Ala Thr Arg Phe Phe Asp Val Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Cys Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Cys 435 440 445 Ser Pro Gly Lys 450 <210> 44 <211> 452 <212> PRT <213> Artificial Sequence <220> <223> Fc region <400> 44 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Asn 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Thr Val Ser Ser Gly Gly Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Gly Thr Thr Ala Leu Ala Thr Arg Phe Phe Asp Val Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Cys 435 440 445 Ser Pro Gly Lys 450 <210> 45 <211> 448 <212> PRT <213> Artificial Sequence <220> <223> Fc region <400> 45 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Asn 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Thr Val Ser Ser Gly Gly Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Gly Thr Thr Ala Leu Ala Thr Arg Phe Phe Asp Val Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Cys 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 <210> 46 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Fc region <400> 46 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Ser Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Ser Ala Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Cys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 47 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Fc region <400> 47 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Ser Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Ser Ala Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Cys 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 48 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Fc region <400> 48 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 1 5 10 15 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 20 25 30 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 35 40 45 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 50 55 60 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Ala Tyr Glu Lys 65 70 75 80 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 85 90 95 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 49 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> LCDR1 <400> 49 Thr Ala Ser Leu Ser Val Ser Ser Thr Tyr Leu His 1 5 10 <210> 50 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> LCDR2 <400> 50 Ser Thr Ser Asn Leu Ala Ser 1 5 <210> 51 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> LCDR3 <400> 51 His Gln Tyr His Arg Ser Pro Leu Thr 1 5 <210> 52 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> LCDR1 <400> 52 Lys Ala Ser Gln Asp Val Gly Thr Ala Val Ala 1 5 10 <210> 53 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> LCDR2 <400> 53 Ser Ala Ser Tyr Arg Ser Thr 1 5 <210> 54 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> LCDR3 <400> 54 Gln His His Tyr Ser Ala Pro Trp Thr 1 5 <210> 55 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> HCDR1 <400> 55 Thr Lys Tyr Gly Val His 1 5 <210> 56 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> HCDR2 <400> 56 Val Lys Trp Ala Gly Gly Ser Thr Asp Tyr Asn Ser Ala Leu Met Ser 1 5 10 15 <210> 57 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> HCDR3 <400> 57 Asp His Arg Asp Ala Met Asp Tyr 1 5 <210> 58 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> HCDR1 <400> 58 Ser Arg Asn Gly Met Ser 1 5 <210> 59 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> HCDR2 <400> 59 Thr Val Ser Ser Gly Gly Ser Tyr Ile Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 60 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> HCDR3 <400> 60 Gln Gly Thr Thr Ala Leu Ala Thr Arg Phe Phe Asp Val 1 5 10 <210> 61 <211> 830 <212> DNA <213> Artificial Sequence <220> <223> IL13Ra2 Ab01 VLVH scFv <400> 61 gctagcccgc caccatgctt ctcctggtga caagccttct gctctgtgag ttaccacacc 60 cagcattcct cctgatccca gacatccaga tgacccagtc cccctcttct ctgtctgcct 120 ctgtgggcga cagagtgacc atcacctgta aggccagtca ggatgtaggt actgctgtag 180 cctggtatca gcagaagcct ggcaaggctc ccaagctgct gatctactcg gcatcctacc 240 ggtccactgg cgtgccttcc agattctccg gctctggctc tggcaccgat ttcaccctga 300 ccatctcctc cctccagcct gaggatttcg ccacctacta ctgccagcac cattatagtg 360 ctccgtggac gtttggcggc ggaacaaagg tggagatcaa gggtggtggt ggttctggcg 420 gcggcggctc cggtggtggt ggttctgagg tgcagctggt ggagtctggc ggcggactgg 480 tgcagcctgg cggctctctg agactgtctt gtgccgcctc cggcttcacc ttcagtagga 540 atggcatgtc ttgggtgagg caggcccctg gcaagggcct ggagtgggtg gccaccgtta 600 gtagtggtgg tagttacatc tactatgcag acagtgtgaa ggggcggttc accatctcca 660 gggacaacgc caagaactcc ctgtacctcc agatgaactc cctgagggcc gaggataccg 720 ccgtgtacta ctgtgccaga caagggacta cggcactagc tacgaggttc ttcgatgtct 780 ggggccaggg caccctggtg accgtgtcct ctgaatctaa gtacggaccg 830 <210> 62 <211> 830 <212> DNA <213> Artificial Sequence <220> <223> IL13Ra2 Ab01 VHVL scFv Ab01-VL sequence Ab01-VH sequence ATG Start codon Gctagc = 5'-NheI restriction site Cggaccg = 3'-RsrII restriction site <400> 62 gctagcccgc caccatgctt ctcctggtga caagccttct gctctgtgag ttaccacacc 60 cagcattcct cctgatccca gaggtgcagc tggtggagtc tggcggcgga ctggtgcagc 120 ctggcggctc tctgagactg tcttgtgccg cctccggctt caccttcagt aggaatggca 180 tgtcttgggt gaggcaggcc cctggcaagg gcctggagtg ggtggccacc gttagtagtg 240 gtggtagtta catctactat gcagacagtg tgaaggggcg gttcaccatc tccagggaca 300 acgccaagaa ctccctgtac ctccagatga actccctgag ggccgaggat accgccgtgt 360 actactgtgc cagacaaggg actacggcac tagctacgag gttcttcgat gtctggggcc 420 agggcaccct ggtgaccgtg tcctctggtg gtggtggttc tggcggcggc ggctccggtg 480 gtggtggttc tgacatccag atgacccagt ccccctcttc tctgtctgcc tctgtgggcg 540 acagagtgac catcacctgt aaggccagtc aggatgtagg tactgctgta gcctggtatc 600 agcagaagcc tggcaaggct cccaagctgc tgatctactc ggcatcctac cggtccactg 660 gcgtgccttc cagattctcc ggctctggct ctggcaccga tttcaccctg accatctcct 720 ccctccagcc tgaggatttc gccacctact actgccagca ccattatagt gctccgtgga 780 cgtttggcgg cggaacaaag gtggagatca aggaatctaa gtacggaccg 830 <210> 63 <211> 815 <212> DNA <213> Artificial Sequence <220> <223> IL13Ra2 Ab02 VLVH scFv <400> 63 gctagcccgc caccatgctt ctcctggtga caagccttct gctctgtgag ttaccacacc 60 cagcattcct cctgatccca gatattcaga tgacccagag cccgagcagc ctgagcgcga 120 gcgtgggcga tcgcgtgacc attacctgca ccgcgagcct gagcgtgagc agcacctatc 180 tgcattggta tcagcagaaa ccgggcaaag cgccgaaact gctgatttat agcaccagca 240 acctggcgag cggcgtgccg agccgcttta gcggcagcgg cagcggcacc gattttaccc 300 tgaccattag cagcctgcag ccggaagatt ttgcgaccta ttattgccat cagtatcatc 360 gcagcccgct gacctttggc ggcggcacca aagtggaaat taaaggtggt ggtggttctg 420 gcggcggcgg ctccggtggt ggtggttctg aagtgcagct ggtggaaagc ggcggcggcc 480 tggtgcagcc gggcggcagc ctgcgcctga gctgcgcggc gagcggcttt acctttacca 540 aatatggcgt gcattgggtg cgccaggcgc cgggcaaagg cctggaatgg gtggcggtga 600 aatgggcggg cggcagcacc gattataaca gcgcgctgat gagccgcttt accattagcc 660 gcgataacgc gaaaaacagc ctgtatctgc agatgaacag cctgcgcgcg gaagataccg 720 cggtgtatta ttgcgcgcgc gatcatcgcg atgcgatgga ttattggggc cagggcaccc 780 tggtgaccgt gagcagcgaa tctaagtacg gaccg 815 <210> 64 <211> 815 <212> DNA <213> Artificial Sequence <220> <223> IL13Ra2 Ab02 VHVL scFv Ab02-VL sequence Ab02-VH sequence ATG Start codon Gctagc = 5'-NheI restriction site Cggaccg = 3'-RsrII restriction site <400> 64 gctagcccgc caccatgctt ctcctggtga caagccttct gctctgtgag ttaccacacc 60 cagcattcct cctgatccca gaagtgcagc tggtggaaag cggcggcggc ctggtgcagc 120 cgggcggcag cctgcgcctg agctgcgcgg cgagcggctt tacctttacc aaatatggcg 180 tgcattgggt gcgccaggcg ccgggcaaag gcctggaatg ggtggcggtg aaatgggcgg 240 gcggcagcac cgattataac agcgcgctga tgagccgctt taccattagc cgcgataacg 300 cgaaaaacag cctgtatctg cagatgaaca gcctgcgcgc ggaagatacc gcggtgtatt 360 attgcgcgcg cgatcatcgc gatgcgatgg attattgggg ccagggcacc ctggtgaccg 420 tgagcagcgg tggtggtggt tctggcggcg gcggctccgg tggtggtggt tctgatattc 480 agatgaccca gagcccgagc agcctgagcg cgagcgtggg cgatcgcgtg accattacct 540 gcaccgcgag cctgagcgtg agcagcacct atctgcattg gtatcagcag aaaccgggca 600 aagcgccgaa actgctgatt tatagcacca gcaacctggc gagcggcgtg ccgagccgct 660 ttagcggcag cggcagcggc accgatttta ccctgaccat tagcagcctg cagccggaag 720 attttgcgac ctattattgc catcagtatc atcgcagccc gctgaccttt ggcggcggca 780 ccaaagtgga aattaaagaa tctaagtacg gaccg 815 <210> 65 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Ab01-VH <400> 65 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Asn 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Thr Val Ser Ser Gly Gly Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Gly Thr Thr Ala Leu Ala Thr Arg Phe Phe Asp Val Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 66 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Ab01 HCDR1 <400> 66 Ser Arg Asn Gly Met Ser 1 5 <210> 67 <211> 17 <212> PRT <213> artificial sequence <220> <223> Ab01 HCDR2 <400> 67 Thr Val Ser Ser Gly Gly Ser Tyr Ile Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 68 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Ab01 HCDR3 <400> 68 Gln Gly Thr Thr Ala Leu Ala Thr Arg Phe Phe Asp Val 1 5 10 <210> 69 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Ab01-VL <400> 69 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Ser Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Ser Ala Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 70 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Ab01 LCDR1 <400> 70 Lys Ala Ser Gln Asp Val Gly Thr Ala Val Ala 1 5 10 <210> 71 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Ab01 LCDR2 <400> 71 Ser Ala Ser Tyr Arg Ser Thr 1 5 <210> 72 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Ab01 LCDR3 <400> 72 Gln His His Tyr Ser Ala Pro Trp Thr 1 5 <210> 73 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Ab02-VH <400> 73 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Lys Tyr 20 25 30 Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Lys Trp Ala Gly Gly Ser Thr Asp Tyr Asn Ser Ala Leu Met 50 55 60 Ser Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp His Arg Asp Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 74 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Ab02 HCDR1 <400> 74 Thr Lys Tyr Gly Val His 1 5 <210> 75 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Ab02 HCDR2 <400> 75 Val Lys Trp Ala Gly Gly Ser Thr Asp Tyr Asn Ser Ala Leu Met Ser 1 5 10 15 <210> 76 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Ab02 HCDR3 <400> 76 Asp His Arg Asp Ala Met Asp Tyr 1 5 <210> 77 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Ab02-VL <400> 77 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Thr Ala Ser Leu Ser Val Ser Ser Thr 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln Tyr His Arg Ser Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 78 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Ab02 LCDR1 <400> 78 Thr Ala Ser Leu Ser Val Ser Ser Thr Tyr Leu His 1 5 10 <210> 79 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Ab02 LCDR2 <400> 79 Ser Thr Ser Asn Leu Ala Ser 1 5 <210> 80 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Ab02 LCDR3 <400> 80 His Gln Tyr His Arg Ser Pro Leu Thr 1 5 <210> 81 <211> 187 <212> PRT <213> Artificial Sequence <220> <223> DHFRdm <400> 81 Met Val Gly Ser Leu Asn Cys Ile Val Ala Val Ser Gln Asn Met Gly 1 5 10 15 Ile Gly Lys Asn Gly Asp Phe Pro Trp Pro Pro Leu Arg Asn Glu Ser 20 25 30 Arg Tyr Phe Gln Arg Met Thr Thr Thr Ser Ser Val Glu Gly Lys Gln 35 40 45 Asn Leu Val Ile Met Gly Lys Lys Thr Trp Phe Ser Ile Pro Glu Lys 50 55 60 Asn Arg Pro Leu Lys Gly Arg Ile Asn Leu Val Leu Ser Arg Glu Leu 65 70 75 80 Lys Glu Pro Pro Gln Gly Ala His Phe Leu Ser Arg Ser Leu Asp Asp 85 90 95 Ala Leu Lys Leu Thr Glu Gln Pro Glu Leu Ala Asn Lys Val Asp Met 100 105 110 Val Trp Ile Val Gly Gly Ser Ser Val Tyr Lys Glu Ala Met Asn His 115 120 125 Pro Gly His Leu Lys Leu Phe Val Thr Arg Ile Met Gln Asp Phe Glu 130 135 140 Ser Asp Thr Phe Phe Pro Glu Ile Asp Leu Glu Lys Tyr Lys Leu Leu 145 150 155 160 Pro Glu Tyr Pro Gly Val Leu Ser Asp Val Gln Glu Glu Lys Gly Ile 165 170 175 Lys Tyr Lys Phe Glu Val Tyr Glu Lys Asn Asp 180 185 <210> 82 <211> 564 <212> DNA <213> Artificial Sequence <220> <223> DHFRdm <400> 82 atggttggtt cgctaaactg catcgtcgct gtgtcccaga acatgggcat cggcaagaac 60 ggggacttcc cctggccacc gctcaggaat gaatccagat atttccagag aatgaccaca 120 acctcttcag tagaaggtaa acagaatctg gtgattatgg gtaagaagac ctggttctcc 180 attcctgaga agaatcgacc tttaaagggt agaattaatt tagttctcag cagagaactc 240 aaggaacctc cacaaggagc tcattttctt tccagaagtc tagatgatgc cttaaaactt 300 actgaacaac cagaattagc aaataaagta gacatggtct ggatagttgg tggcagttct 360 gtttataagg aagccatgaa tcacccaggc catcttaaac tatttgtgac aaggatcatg 420 caagactttg aaagtgacac gttttttcca gaaattgatt tggagaaata taaacttctg 480 ccagaatacc caggtgttct ctctgatgtc caggaggaga aaggcattaa gtacaaattt 540 gaagtatatg agaagaatga ttaa 564 <210> 83 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> GMCSFss Leader <400> 83 atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60 atccca 66 <210> 84 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> Ab01-VL <400> 84 gacatccaga tgacccagtc cccctcttct ctgtctgcct ctgtgggcga cagagtgacc 60 atcacctgta aggccagtca ggatgtaggt actgctgtag cctggtatca gcagaagcct 120 ggcaaggctc ccaagctgct gatctactcg gcatcctacc ggtccactgg cgtgccttcc 180 agattctccg gctctggctc tggcaccgat ttcaccctga ccatctcctc cctccagcct 240 gaggatttcg ccacctacta ctgccagcac cattatagtg ctccgtggac gtttggcggc 300 ggaacaaagg tggagatcaa g 321 <210> 85 <211> 366 <212> DNA <213> Artificial Sequence <220> <223> Ab01-VH <400> 85 gaggtgcagc tggtggagtc tggcggcgga ctggtgcagc ctggcggctc tctgagactg 60 tcttgtgccg cctccggctt caccttcagt aggaatggca tgtcttgggt gaggcaggcc 120 cctggcaagg gcctggagtg ggtggccacc gttagtagtg gtggtagtta catctactat 180 gcagacagtg tgaaggggcg gttcaccatc tccagggaca acgccaagaa ctccctgtac 240 ctccagatga actccctgag ggccgaggat accgccgtgt actactgtgc cagacaaggg 300 actacggcac tagctacgag gttcttcgat gtctggggcc agggcaccct ggtgaccgtg 360 tcctct 366 <210> 86 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> Ab02-VL <400> 86 gatattcaga tgacccagag cccgagcagc ctgagcgcga gcgtgggcga tcgcgtgacc 60 attacctgca ccgcgagcct gagcgtgagc agcacctatc tgcattggta tcagcagaaa 120 ccgggcaaag cgccgaaact gctgatttat agcaccagca acctggcgag cggcgtgccg 180 agccgcttta gcggcagcgg cagcggcacc gattttaccc tgaccattag cagcctgcag 240 ccggaagatt ttgcgaccta ttattgccat cagtatcatc gcagcccgct gacctttggc 300 ggcggcacca aagtggaaat taaa 324 <210> 87 <211> 348 <212> DNA <213> Artificial Sequence <220> <223> Ab02-VH <400> 87 gaagtgcagc tggtggaaag cggcggcggc ctggtgcagc cgggcggcag cctgcgcctg 60 agctgcgcgg cgagcggctt tacctttacc aaatatggcg tgcattgggt gcgccaggcg 120 ccgggcaaag gcctggaatg ggtggcggtg aaatgggcgg gcggcagcac cgattataac 180 agcgcgctga tgagccgctt taccattagc cgcgataacg cgaaaaacag cctgtatctg 240 cagatgaaca gcctgcgcgc ggaagatacc gcggtgtatt attgcgcgcg cgatcatcgc 300 gatgcgatgg attattgggg ccagggcacc ctggtgaccg tgagcagc 348 <210> 88 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> (Gly-Gly-Gly-Ser)x3 linker <400> 88 ggtggtggtg gttctggcgg cggcggctcc ggtggtggtg gttct 45 <210> 89 <211> 732 <212> DNA <213> Artificial Sequence <220> <223> Ab01 scFv VLVH <400> 89 gacatccaga tgacccagtc cccctcttct ctgtctgcct ctgtgggcga cagagtgacc 60 atcacctgta aggccagtca ggatgtaggt actgctgtag cctggtatca gcagaagcct 120 ggcaaggctc ccaagctgct gatctactcg gcatcctacc ggtccactgg cgtgccttcc 180 agattctccg gctctggctc tggcaccgat ttcaccctga ccatctcctc cctccagcct 240 gaggatttcg ccacctacta ctgccagcac cattatagtg ctccgtggac gtttggcggc 300 ggaacaaagg tggagatcaa gggtggtggt ggttctggcg gcggcggctc cggtggtggt 360 ggttctgagg tgcagctggt ggagtctggc ggcggactgg tgcagcctgg cggctctctg 420 agactgtctt gtgccgcctc cggcttcacc ttcagtagga atggcatgtc ttgggtgagg 480 caggcccctg gcaagggcct ggagtgggtg gccaccgtta gtagtggtgg tagttacatc 540 tactatgcag acagtgtgaa ggggcggttc accatctcca gggacaacgc caagaactcc 600 ctgtacctcc agatgaactc cctgagggcc gaggataccg ccgtgtacta ctgtgccaga 660 caagggacta cggcactagc tacgaggttc ttcgatgtct ggggccaggg caccctggtg 720 accgtgtcct ct 732 <210> 90 <211> 732 <212> DNA <213> Artificial Sequence <220> <223> Ab01 scFv VHVL <400> 90 gaggtgcagc tggtggagtc tggcggcgga ctggtgcagc ctggcggctc tctgagactg 60 tcttgtgccg cctccggctt caccttcagt aggaatggca tgtcttgggt gaggcaggcc 120 cctggcaagg gcctggagtg ggtggccacc gttagtagtg gtggtagtta catctactat 180 gcagacagtg tgaaggggcg gttcaccatc tccagggaca acgccaagaa ctccctgtac 240 ctccagatga actccctgag ggccgaggat accgccgtgt actactgtgc cagacaaggg 300 actacggcac tagctacgag gttcttcgat gtctggggcc agggcaccct ggtgaccgtg 360 tcctctggtg gtggtggttc tggcggcggc ggctccggtg gtggtggttc tgacatccag 420 atgacccagt ccccctcttc tctgtctgcc tctgtgggcg acagagtgac catcacctgt 480 aaggccagtc aggatgtagg tactgctgta gcctggtatc agcagaagcc tggcaaggct 540 cccaagctgc tgatctactc ggcatcctac cggtccactg gcgtgccttc cagattctcc 600 ggctctggct ctggcaccga tttcaccctg accatctcct ccctccagcc tgaggatttc 660 gccacctact actgccagca ccattatagt gctccgtgga cgtttggcgg cggaacaaag 720 gtggagatca ag 732 <210> 91 <211> 717 <212> DNA <213> Artificial Sequence <220> <223> Ab02 scFv VLVH <400> 91 gatattcaga tgacccagag cccgagcagc ctgagcgcga gcgtgggcga tcgcgtgacc 60 attacctgca ccgcgagcct gagcgtgagc agcacctatc tgcattggta tcagcagaaa 120 ccgggcaaag cgccgaaact gctgatttat agcaccagca acctggcgag cggcgtgccg 180 agccgcttta gcggcagcgg cagcggcacc gattttaccc tgaccattag cagcctgcag 240 ccggaagatt ttgcgaccta ttattgccat cagtatcatc gcagcccgct gacctttggc 300 ggcggcacca aagtggaaat taaaggtggt ggtggttctg gcggcggcgg ctccggtggt 360 ggtggttctg aagtgcagct ggtggaaagc ggcggcggcc tggtgcagcc gggcggcagc 420 ctgcgcctga gctgcgcggc gagcggcttt acctttacca aatatggcgt gcattgggtg 480 cgccaggcgc cgggcaaagg cctggaatgg gtggcggtga aatgggcggg cggcagcacc 540 gattataaca gcgcgctgat gagccgcttt accattagcc gcgataacgc gaaaaacagc 600 ctgtatctgc agatgaacag cctgcgcgcg gaagataccg cggtgtatta ttgcgcgcgc 660 gatcatcgcg atgcgatgga ttattggggc cagggcaccc tggtgaccgt gagcagc 717 <210> 92 <211> 717 <212> DNA <213> Artificial Sequence <220> <223> Ab02 scFv VHVL <400> 92 gaagtgcagc tggtggaaag cggcggcggc ctggtgcagc cgggcggcag cctgcgcctg 60 agctgcgcgg cgagcggctt tacctttacc aaatatggcg tgcattgggt gcgccaggcg 120 ccgggcaaag gcctggaatg ggtggcggtg aaatgggcgg gcggcagcac cgattataac 180 agcgcgctga tgagccgctt taccattagc cgcgataacg cgaaaaacag cctgtatctg 240 cagatgaaca gcctgcgcgc ggaagatacc gcggtgtatt attgcgcgcg cgatcatcgc 300 gatgcgatgg attattgggg ccagggcacc ctggtgaccg tgagcagcgg tggtggtggt 360 tctggcggcg gcggctccgg tggtggtggt tctgatattc agatgaccca gagcccgagc 420 agcctgagcg cgagcgtggg cgatcgcgtg accattacct gcaccgcgag cctgagcgtg 480 agcagcacct atctgcattg gtatcagcag aaaccgggca aagcgccgaa actgctgatt 540 tatagcacca gcaacctggc gagcggcgtg ccgagccgct ttagcggcag cggcagcggc 600 accgatttta ccctgaccat tagcagcctg cagccggaag attttgcgac ctattattgc 660 catcagtatc atcgcagccc gctgaccttt ggcggcggca ccaaagtgga aattaaa 717 <210> 93 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> S spacer <400> 93 gaatctaagt acggaccgcc ctgcccccct tgccct 36 <210> 94 <211> 84 <212> DNA <213> Artificial Sequence <220> <223> CD28tm <400> 94 atgttctggg tgctggtggt ggtcggaggc gtgctggcct gctacagcct gctggtcacc 60 gtggccttca tcatcttttg ggtg 84 <210> 95 <211> 126 <212> DNA <213> Artificial Sequence <220> <223> 41-BB <400> 95 aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60 actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120 gaactg 126 <210> 96 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> CD3? <400> 96 cgggtgaagt tcagcagaag cgccgacgcc cctgcctacc agcagggcca gaatcagctg 60 tacaacgagc tgaacctggg cagaagggaa gagtacgacg tcctggataa gcggagaggc 120 cgggaccctg agatgggcgg caagcctcgg cggaagaacc cccaggaagg cctgtataac 180 gaactgcaga aagacaagat ggccgaggcc tacagcgaga tcggcatgaa gggcgagcgg 240 aggcggggca agggccacga cggcctgtat cagggcctgt ccaccgccac caaggatacc 300 tacgacgccc tgcacatgca ggccctgccc ccaagg 336 <210> 97 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> T2A <400> 97 ctcgagggcg gcggagaggg cagaggaagt cttctaacat gcggtgacgt ggaggagaat 60 cccggcccta gg 72 <210> 98 <211> 1005 <212> DNA <213> Artificial Sequence <220> <223> EGFRt <400> 98 cgcaaagtgt gtaacggaat aggtattggt gaatttaaag actcactctc cataaatgct 60 acgaatatta aacacttcaa aaactgcacc tccatcagtg gcgatctcca catcctgccg 120 gtggcattta ggggtgactc cttcacacat actcctcctc tggatccaca ggaactggat 180 attctgaaaa ccgtaaagga aatcacaggg tttttgctga ttcaggcttg gcctgaaaac 240 aggacggacc tccatgcctt tgagaaccta gaaatcatac gcggcaggac caagcaacat 300 ggtcagtttt ctcttgcagt cgtcagcctg aacataacat ccttgggatt acgctccctc 360 aaggagataa gtgatggaga tgtgataatt tcaggaaaca aaaatttgtg ctatgcaaat 420 acaataaact ggaaaaaact gtttgggacc tccggtcaga aaaccaaaat tataagcaac 480 agaggtgaaa acagctgcaa ggccacaggc caggtctgcc atgccttgtg ctcccccgag 540 ggctgctggg gcccggagcc cagggactgc gtctcttgcc ggaatgtcag ccgaggcagg 600 gaatgcgtgg acaagtgcaa ccttctggag ggtgagccaa gggagtttgt ggagaactct 660 gagtgcatac agtgccaccc agagtgcctg cctcaggcca tgaacatcac ctgcacagga 720 cggggaccag acaactgtat ccagtgtgcc cactacattg acggccccca ctgcgtcaag 780 acctgcccgg caggagtcat gggagaaaac aacaccctgg tctggaagta cgcagacgcc 840 ggccatgtgt gccacctgtg ccatccaaac tgcacctacg gatgcactgg gccaggtctt 900 gaaggctgtc caacgaatgg gcctaagatc ccgtccatcg ccactgggat ggtgggggcc 960 ctcctcttgc tgctggtggt ggccctgggg atcggcctct tcatg 1005 <210> 99 <211> 564 <212> DNA <213> Artificial Sequence <220> <223> DHFRdm <400> 99 atggttggtt cgctaaactg catcgtcgct gtgtcccaga acatgggcat cggcaagaac 60 ggggacttcc cctggccacc gctcaggaat gaatccagat atttccagag aatgaccaca 120 acctcttcag tagaaggtaa acagaatctg gtgattatgg gtaagaagac ctggttctcc 180 attcctgaga agaatcgacc tttaaagggt agaattaatt tagttctcag cagagaactc 240 aaggaacctc cacaaggagc tcattttctt tccagaagtc tagatgatgc cttaaaactt 300 actgaacaac cagaattagc aaataaagta gacatggtct ggatagttgg tggcagttct 360 gtttataagg aagccatgaa tcacccaggc catcttaaac tatttgtgac aaggatcatg 420 caagactttg aaagtgacac gttttttcca gaaattgatt tggagaaata taaacttctg 480 ccagaatacc caggtgttct ctctgatgtc caggaggaga aaggcattaa gtacaaattt 540 gaagtatatg agaagaatga ttaa 564 <210> 100 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> M spacer <400> 100 gaatctaagt acggaccgcc ctgcccccct tgccctggcc agcctagaga accccaggtg 60 tacaccctgc ctcccagcca ggaagagatg accaagaacc aggtgtccct gacctgcctg 120 gtcaaaggct tctaccccag cgatatcgcc gtggaatggg agagcaacgg ccagcccgag 180 aacaactaca agaccacccc ccctgtgctg gacagcgacg gcagcttctt cctgtactcc 240 cggctgaccg tggacaagag ccggtggcag gaaggcaacg tcttcagctg cagcgtgatg 300 cacgaggccc tgcacaacca ctacacccag aagtccctga gcctgagcct gggcaag 357 <210> 101 <211> 685 <212> DNA <213> Artificial Sequence <220> <223> L spacer <400> 101 atctaagtac ggaccgccct gccccccttg ccctgccccc gagttcgacg gcggacccag 60 cgtgttcctg ttccccccca agcccaagga caccctgatg atcagccgga cccccgaggt 120 gacctgcgtg gtggtggacg tgagccagga agatcccgag gtccagttca attggtacgt 180 ggacggcgtg gaagtgcaca acgccaagac caagcccaga gaggaacagt tccagagcac 240 ctaccgggtg gtgtctgtgc tgaccgtgct gcaccaggac tggctgaacg gcaaagaata 300 caagtgcaag gtgtccaaca agggcctgcc cagcagcatc gaaaagacca tcagcaaggc 360 caagggccag cctcgcgagc cccaggtgta caccctgcct ccctcccagg aagagatgac 420 caagaaccag gtgtccctga cctgcctggt gaagggcttc taccccagcg acatcgccgt 480 ggagtgggag agcaacggcc agcctgagaa caactacaag accacccctc ccgtgctgga 540 cagcgacggc agcttcttcc tgtacagccg gctgaccgtg gacaagagcc ggtggcagga 600 aggcaacgtc tttagctgca gcgtgatgca cgaggccctg cacaaccact acacccagaa 660 gagcctgagc ctgtccctgg gcaag 685

Claims (66)

A ligand binding domain that binds and/or interacts with the IL-13 alpha 2 (IL13Rα2) receptor;
A polypeptide spacer between the ligand binding domain and the transmembrane domain;
Transmembrane domain; And
Intracellular signaling region
Chimeric antigen receptor comprising
Nucleic acid encoding. The method of claim 1, wherein the ligand binding domain is
Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 20, or a conservative variant thereof;
Heavy chain complementarity determining region 2 (CDR2) having the amino acid sequence of SEQ ID NO: 21, or a conservative variant thereof; And/or
Heavy chain complementarity determining region 3 (CDR3) having the amino acid sequence of SEQ ID NO: 22, or a conservative variant thereof
A nucleic acid comprising a. The method of claim 1 or 2, wherein the ligand binding domain is
Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 23, or a conservative variant thereof;
Light chain complementarity determining region 2 (CDR2) having the amino acid sequence of SEQ ID NO: 24, or a conservative variant thereof; And/or
Light chain complementarity determining region 3 (CDR3) having the amino acid sequence of SEQ ID NO: 25, or a conservative variant thereof
A nucleic acid comprising a. The nucleic acid according to any one of claims 1 to 3, wherein the ligand binding domain comprises a heavy chain variable (VH) domain comprising a polypeptide having at least 90% identity to the amino acid sequence of SEQ ID NO:18. The nucleic acid according to any one of claims 1 to 4, wherein the ligand binding domain comprises a light chain variable (VL) domain comprising a polypeptide having at least 90% identity to the amino acid sequence of SEQ ID NO:19. The method of any one of claims 1 to 5, wherein the ligand binding domain is
Heavy chain CDR1 having the amino acid sequence of SEQ ID NO:20;
Heavy chain CDR2 having the amino acid sequence of SEQ ID NO:21; And/or
Heavy chain CDR3 having the amino acid sequence of SEQ ID NO:22
A nucleic acid comprising a. The method of any one of claims 1 to 6, wherein the ligand binding domain is
Light chain CDR1 having the amino acid sequence of SEQ ID NO:23;
Light chain CDR2 having the amino acid sequence of SEQ ID NO:24; And/or
Light chain CDR3 having the amino acid sequence of SEQ ID NO:25
A nucleic acid comprising a. The nucleic acid according to any one of claims 1 to 7, wherein the ligand binding domain comprises a VH domain comprising a polypeptide having the amino acid sequence of SEQ ID NO:18. The nucleic acid according to any one of claims 1 to 8, wherein the ligand binding domain comprises a VL domain having the amino acid sequence of SEQ ID NO:19. The nucleic acid according to any one of claims 1 to 9, wherein the ligand binding domain is an antibody fragment, such as an antigen-binding fragment. The nucleic acid according to any one of claims 1 to 10, wherein the ligand binding domain is a single chain variable fragment (scFv). 12. The nucleic acid of any one of claims 1 to 11, wherein the scFv comprises a VL-VH orientation. 13. The nucleic acid of any one of claims 1-12, wherein the single chain variable fragment (scFv) comprises a sequence having at least 95% identity to the nucleotide sequence of SEQ ID NO:61. The nucleic acid according to any one of claims 1 to 13, wherein the single chain variable fragment (scFv) comprises the nucleotide sequence of SEQ ID NO:61. 15. The nucleic acid according to any one of claims 1 to 14, wherein the spacer comprises an amino acid sequence of X ₁ PPX ₂ P. 16. The nucleic acid according to any one of claims 1 to 15, wherein the spacer region comprises a portion of the hinge region of a human antibody or a modified variant thereof. 17. The nucleic acid according to any one of claims 1 to 16, wherein the spacer is 15 or less amino acids, but 1 or 2 or more amino acids. The nucleic acid of any one of claims 1-17, wherein the spacer comprises, consists of, or consists essentially of a sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:09. 19. The nucleic acid of any one of claims 1-18, wherein the spacer comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:09 or a conservative substitution thereof. The nucleic acid according to any one of the preceding claims, wherein the spacer comprises, consists of, or consists essentially of an IgG4 hinge spacer (S). 21. A nucleic acid according to any one of the preceding claims, wherein the spacer comprises, consists of, or consists essentially of an IgG4 hinge-CH3 spacer (M). The nucleic acid according to any one of the preceding claims, wherein the spacer comprises, consists of, or consists essentially of an IgG4 hinge-CH2 (L234D, N297A)-CHE spacer (L). The method according to any one of claims 1 to 22, wherein the intracellular signaling region is CD27, CD28, 4-1BB, OX-40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, Nucleic acid comprising primary and co-stimulatory signaling domains optionally containing all or part of CD3 zeta in combination with a co-stimulatory domain selected from the group consisting of the signaling domains of NKG2C and B7-H3 and combinations thereof. The nucleic acid of claim 23, wherein the intracellular signaling region comprises a signaling portion of CD3 zeta and a signaling portion of 4-1BB. 25. The nucleic acid of any one of claims 1-24, wherein the transmembrane domain comprises a CD28 transmembrane domain (CD28tm). 26. The nucleic acid according to any one of claims 1 to 25, further comprising a nucleic acid encoding a marker sequence. 27. The nucleic acid of claim 26, wherein the marker sequence is a truncated receptor, optionally EGFRt, HER2t or CD19t. 28. The nucleic acid of any one of claims 1-27, further comprising a dihydrofolate reductase transgene configured for methotrexate selection. 29. The nucleic acid of claim 28, wherein the dihydrofolate reductase transgene is a dihydrofolate reductase double mutant (DHFRdm). 30. The nucleic acid of claim 29, wherein the dihydrofolate reductase double mutant comprises amino acid mutations of L22F and F31S. An expression vector comprising the nucleic acid of any one of claims 1 to 30. The expression vector according to claim 31, which is a viral vector. The expression vector according to claim 30 or 31, which is a lentiviral or adenovirus vector. A chimeric antigen receptor (CAR) polypeptide encoded by the nucleic acid of any one of claims 1 to 30. A host cell comprising the nucleic acid of any one of claims 1 to 30. 36. The host cell of claim 35, which is a CD8+ T cytotoxic lymphocyte cell selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, and bulk CD8+ T cells. 37. The host cell of claim 36, wherein the CD8+ cytotoxic T lymphocyte cells are central memory T cells and the central memory T cells are positive for CD45RO+, CD62L+ and CD8+. 36. The host cell of claim 35, which is a CD4+ T helper lymphocyte cell selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells. 39. The host cell of claim 38, wherein the CD4+ helper lymphocyte cells are naive CD4+ T cells and the naive CD4+ T cells are positive for CD45RA+, CD62L+ and CD4+ and negative for CD45RO. 40. The host cell of any one of claims 35 to 39, which is a precursor T cell. 41. The host cell according to any one of claims 35 to 40, which is a hematopoietic stem cell. A pharmaceutical composition comprising the host cell of any one of claims 35 to 41 and a pharmaceutically acceptable excipient. Introducing the nucleic acid of any one of claims 1 to 30 into a cell; And
Culturing the cells in conditions sufficient for the cells to expand in the presence of an anti-CD3 antibody and/or anti CD28 antibody and at least one homeostatic cytokine; And
Selecting a cell as a selection reagent; Wherein the selection reagent is configured to selectively enrich cells transduced with nucleic acids or vectors.
A method for producing a host cell according to any one of claims 35 to 41 comprising a. Introducing the nucleic acid of any one of claims 1 to 30 into a cell; And
Culturing the cells in conditions sufficient for the cells to expand in the presence of an anti-CD3 antibody and/or anti CD28 antibody and at least one homeostatic cytokine; And
Selecting a cell as a selection reagent; Wherein the selection reagent is configured to selectively enrich cells transduced with nucleic acids or vectors.
A method for producing a host cell comprising a. 45. The method of claim 43 or 44, wherein the cell is a lymphocyte. 46. The method of claim 45, wherein the lymphocyte has a CD45RA-, CD45RO+ and CD62L+ phenotype. 47. The method of claim 45 or 46, wherein the lymphocyte is CD8+ or CD4+. 48. The method of any one of claims 43-47, wherein the reagent of choice is methotrexate. 49. The method of any one of claims 43-48, wherein the cytokine is IL-15, Il-7 and/or Il-21. 50. The method of any one of claims 43-49, further comprising introducing a second nucleic acid into the host cell, wherein the second nucleic acid encodes a marker protein. 51. The method of claim 50, wherein the marker protein is EGFRt. The host cell of any one of claims 35 to 41 for use in a medicament or for use in the treatment or inhibition of cancer or solid tumors expressing the IL-13α2 receptor. 53. The use according to claim 52, wherein the cancer comprises brain cancer. 54. The use according to claim 52 or 53, wherein the cancer is a glioma or glioblastoma tumor. 55. The use according to claim 54, wherein the cancer is glioblastoma polymorphic (GBM). 55. The use according to claim 54, wherein the cancer is a glioma. A method of treating, inhibiting or alleviating cancer in a subject, comprising administering the host cell of any one of claims 35 to 41 to a subject in need thereof. 58. The method of claim 57, wherein the cancer is an IL13Rα-positive malignant tumor. 59. The method of claim 57 or 58, wherein the cancer is brain cancer. 60. The method of any one of claims 57-59, wherein the cancer is a glioma or glioblastoma tumor. 61. The method of any one of claims 57-60, wherein the cancer is a glioma. 61. The method of any one of claims 57-60, wherein the cancer is glioblastoma polymorphic (GBM). 63. The method of any one of claims 57-62, further comprising administering an additional therapy selected from chemotherapy and radiation therapy. The method of claim 63, wherein the chemotherapeutic drug is electrochemotherapy, an alkylating agent, an antimetabolite (e.g. 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine ( Geloda®), cladribine, cloparabine, cytarabine (Ara-C®), phloxuridine, fludarabine, gemcitabine (Gemza®), hydroxyurea, methotrexate, pemetrexide (Alymta®) , Pentostatin, and thioguanine), anti-tumor antibiotics, topoisomerase inhibitors, mitosis inhibitors, corticosteroids, DNA intercalating agents, or checkpoint inhibitors (checkpoint kinase CHK1, CHK2). 65. The method of any one of claims 57-64, wherein the subject is a mammal. 66. The method of any one of claims 57-65, wherein the subject is a human.

Images